Methods for detecting teneurin signalling and related screening methods

ABSTRACT

Methods are provided for detecting teneurin signaling by determining the presence and/or amount of a cleaved teneurin product, and correlating the presence and/or amount of the cleaved teneurin product with teneurin signalling. The methods can be used for screening possible anti-tumour agents or agents that modulate neuronal differentiation. Test substances are assayed for their ability to increase or decrease the presence of the cytoplasmic domain of teneurin or of its respective targets, such as ponsin, PML an zic.

FIELD OF THE INVENTION

The present invention relates to methods of detecting teneurinsignalling and methods for screening agents effective in modulatingteneurin signalling with applications In the fields of neurobiology andoncology. In particular, agents affecting teneurin signalling may beuseful as anti-tumour and/or anti-tumourigenic agents, or for modulatingneuronal differentiation, development or regeneration.

BACKGROUND OF THE INVENTION

Teneurins are a family of type II transmembrane proteins originallydiscovered in Drosophila. The first member was Ten-a (Baumgartner andChiquet-Ehrismann (1993) Mech Dev 40:165-76; Minet and Chiquet-Ehrismann(2000) Gene 257: 87-97), which was found in a search for Drosophilahomologues of tenascins and shares with this protein family the sametype of EGF-like repeats. The second member of the teneurin family,Drosophila Ten-m/Odd oz (Odz; Baumgartner et al. (1994) Embo J 13:3728-40) is expressed in seven stripes during the blastoderm stage ofearly embryos. Mutational analysis showed that ten-n/odz is a member ofthe “pair-rule” gene family and encodes for an extracellular proteinhaving a central role in determining the segmentation of the embryo. Theexpression pattern in the developing embryos and the adult fly suggestfurther important activities of this protein in later developmentalprocesses since its presence often coincides with locations ofmorphogenetic cell movements, in particular, during gastrulation, thedevelopment of the tracheal system, on pioneering axons and in thedeveloping eye (Baumgartner et al. (1994) Embo J 13: 3728-40); (Levineet al. (1994) Cell 77: 587-98); (Levine et al. (1997) Dev Dyn 209:1-14).

Vertebrates contain four ten-a/ten-m homologues, termed ten-m1-4(Oohashi et al. (1999) J Cell Biol 145: 563-77), odz1-4 (Ben-Zur et al.(2000) Dev Biol 217: 107-20), or teneurin 1-4 (Minet andChiquet-Ehrismann (2000) Gene 257: 87-97), respectively. A recent studypresented two new members of this family of proteins in chicken, namely,teneurin-1 and teneurin-2. Both the Drosophila and chicken proteins areexpressed In early stages of embryonic development, In particular, inthe developing nervous system and suggest an interaction or teneurin-2with the cytoskeleton. Expression of recombinant teneurin-2 in aneuroblastoma cell line appears to lead to filopodia formation andenlarged growth cones (Rubin et al. (1999) Dev Biol 216: 195-209).

Mouse DOC4, the first vertebrate member of the teneurin family, wasidentified in a screen for proteins that were expressed in response toperturbation of protein folding in the endoplasmic reticulum (Wang etal. (1998) Embo J 17 3619-30). Mouse DOC4 and all other vertebrateteneurins identified—namely, the mouse teneurins ten-m1-4/odz1-4(Oohashi et al. (1999) J Cell Biol 145: 563-7);(Ben-Zur et al. (2000)Dev Biol 217: 107-20), the rat teneurin-2 orthologue, neurestin (Otakiand Firestein (1999) Dev Biol 212: 165-81), the chicken teneurins-1, -2and -4 (Minet et al. (1999) J Cell Sci 112: 2019-32); (Rubin et al.(1999) Dev Biol 216:195-209); (Tucker et al. (2000) Mech Dev98, 187-91);(Tucker et al. (2001) Dev Dyn 220: 27-39) and the zebrafish Ten-m3 andTen-m4 (Mieda et al. (1999) Mech Dev 87, 223-7) - were found to beprominently expressed in specific regions of the central nervous system.

Except for transcript localization by in situ hybridization in thenervous system and the possible involvement of teneurin-2 in theorganization of the actin cytoskeleton in neuroblastoma cells, verylittle is known about the distribution and function of teneurins.Teneurins are known to be transmembrane proteins and are postulated tobe involved in signal transduction. It has been suggested that teneurinscould function as receptor proteins transmitting signals to the cellinterior upon homo- or heterophilic binding of a ligand or as amembrane-bound ligand. Teneurin-2 is thought to be cleaved at the cellmembrane either releasing a large part of the extracellular domain fromthe cell membrane, which could act as a soluble ligand or cleavage couldoccur after ligand binding and result in signal transduction (Rubin etal., Developmental Biology, 216,195-209, 1999).

One potential scenario by which transmembrane proteins can fulfil theirrole as signalling molecules is by a mechanism recently described asregulated intramembrane proteolysis (RIP; reviewed in Brown et al.(2000) Cell 100, 391-98). RIP involves at least two cleavage steps inand at the membrane resulting in a soluble cytoplasmic part, which Istranslocated to the nucleus where it participates in transcription(Ebinu and Yankner (2002) Neuron 34(4):499-502. RIP is known to controldiverse cellular and developmental processes. It is well known, however,that transmembrane proteins can initiate signal transduction byalternative mechanisms, such as ligand binding, receptor binding, andsignalling through kinase/phosphatase cascades, etc. Indeed, Drosophilaten-m was postulated to modulate the activity of the Drosophilapair-rule gene, Odd-paired (opa) protein via a signal transductioncascade (Baumgartner et al., 1994, EMBO J. 13: 3728-3740), although asimilarity between the intracellular cleavages of Odz have been likenedto those of notch (Dgany and Wides, Biochem J. (2992) 363:633-643).Netherless, no evidence exists that any portion of Odz translocates tothe nucleus. Furthermore, the extracellular domain of mouse Ten m1 wasfound to exhibit homophilic binding and thereby inittate a signaltransduction pathway (Oohashi et al., 1999).

Zic genes (Zic1-4) encode zinc finger proteins homologous to opa, andare expressed in both the developing and mature CNS. In vertebrateneural development, they are generally expressed in the dorsal neuraltube. A similar zinc finger gene, Opr, has also been described in mice(Furushima et al. (2000) Mech. of Development 98, 161-64). Zic1 controlsthe expansion of neuronal precursors by inhibiting the progression ofneuronal differentiation. Notch-mediated inhibition of neuronaldifferentiation is likely to act downstream of Zic genes since Notch1 isupregulated in Zic1-overexpressing spinal cords in both the mouse andthe chick (Aruga et al. (2002) Dev Biol. 244(2):329-41). Reducedexpression of Zic2 in mice results in spina bifida andholoprosencephaly. The disruption of Zic1, a strong homolog of Zic2 thathas an overlapping expression pattern, results in cerebellarmalformation. It has been shown that Zic2 and Zic1 cooperatively controlcerebellar development by regulating neuronal differentiation (Aruga etal. (2002) J Neurosci 22(1):218-25).

The ponsin (also known as SH3P12, CAP or FLAF2) gene encodes a proteinbelonging to the Ponsin/ArgBP2/venexin family. All members of thisfamily contain three SH3 (src homology 3 region) domains. It is throughthese SH3 domains that Ponsin protein interacts with Vinculin, anF-actin binding protein, at cell-cell and cell-matrix adherens junctionsor with Afadin at Zonula adherens (Mandai et al. (1999) J. Cell Biol.144: 1001 -1017). Ponsin also directly interacts with the non-receptorfocal adhesion tyrosine kinase p125 FAK (Ribon et al. (1 998a) J. Biol.Chem. 273: 4073-4080). Several splice variants of Ponsin mRNA exist,which are specifically up-regulated by p53 expression in EB-1 cells andby adriamycin treatment of TK6 cells (Kostic et al. (2000) Oncogene19(35):3978-87). Loss of wild type p53 function in colon tumourscoincide with migration of the tumour mass across the basement membrane.

PML (a protein named for its prevalence in promyelocytic leukemia) is anuclear protein that controls apoptosis, cell proliferation andsenescence and it is believed to be a zinc finger transcription factor.The PML gene, involved in the chromosomal translocation of acutepromyelocytic leukemia (APL), encodes a protein which Is located Innuclear substructures called PML-bodies and which plays a role in tumoursuppression (reviewed in Seeler et al. (1999) Curr Opin Genet Dev.9(3):362-7; or Salomoni and Pandolfi (2002) Cell 108, 165-70).

A need exists to find signalling pathways and genes that are involved inthe development of neuropathological, neurodevelopmental, andneurodegenerative diseases or conditions as well as in other therapeuticareas, such as cancer, to more accurately and effectively diagnose andtreat these diseases. A more complete delineation of the teneurinsignalling pathway and identification of the pathway's componentsprovided by the present invention meets this need.

SUMMARY OF THE INVENTION

In accordance with a first aspect of the invention a method fordetecting teneurin signalling is provided, which method comprisesproviding (i) a teneurin or a fragment thereof, which fragment comprisesat least a portion of the N-terminal domain of teneurin and at least aportion of the C-terminal domain of teneurin, and (ii) a cellularcomponent that cleaves the teneurin; determining the presence and/oramount of a cleaved teneurin product associated with the signalling; andcorrelating the presence and/or amount of the cleaved teneurin productwith teneurin signalling. The cleaved teneurin product comprises thecytoplasmic domain of teneurin or a fragment thereof and may be presentwithout limitation in tumour cells containing PML nuclear bodies or inneurons. In one embodiment teneurin is teneurin-1, in another embodimentit is teneurin-2, teneurin-3, or teneurin-4.

The method of the invention for detecting teneurin signalling furthercomprises labelling teneurin with a detectable tag or label. In oneaspect, the labelled teneurin is recombinant. In another embodiment ofthe present invention, the cleaved teneurin product comprises adetectable tag or label. Said tag or label may be detectedphotometrically and may be without limitation GFP (green fluorescenceprotein) or YFP (yellow fluorescence protein).

In one aspect, the determination of the presence and/or amount of acleaved teneurin product associated with the signalling is qualitative.In a further aspect, the determination is quantitative (i.e., the amountof cleaved teneurin is detected not merely its presence).

In a further embodiment, a method is provided for detecting teneurinsignalling further comprising expressing tagged teneurin from nucleicacid encoding therefor introduced into a cell or a progenitor thereof,wherein said tag may comprise a DNA binding domain which binds tonucleic acid comprising regulatory sequences operably linked to areporter gene. Said DNA binding domain may comprise without limitationGAL 4 DNA binding domain. In another embodiment, said tag furthercomprises a NFκB domain. The present invention further comprises nucleicacid encoding for tagged teneurin which is stably expressed in a cell,e.g. a tumour cell or a neuron.

In a further aspect, the present invention provides a method fordetecting teneurin signalling wherein the cleaved teneurin productregulates expression or activity of a cellular target which itself is amodulator of cell proliferation or neuronal differentiation, e.g. PML,zic, ponsin, p53 and myc, or wherein the cleaved teneurin producttargets to the nucleus. Said cellular target of the cleaved teneurinproduct may be detected by expression array analysis.

Also provided is a method for detecting teneurin signalling wherein thepresence and/or amount of the detectable cleaved teneurin product iscorrelated to a particular disease. This method can be performed fordetermining whether a cleaved teneurin product is involved in cellproliferation or neuronal differentiation.

In one embodiment, an in vitro method of diagnosis of a neuropathologyor cell pathology which affects teneurin-mediated signalling is providedwhich comprises performing the hereinabove described methods of theinvention on a cell which has been extracted from an animal which it isdesired to diagnose.

In another embodiment, the use of a detectable cleaved teneurin productassociated with teneurin signalling in an in vivo method of diagnosis ofa neuropathology or cell pathology which affects teneurin signalling isprovided, which method comprises performing a method of the invention ashereinabove described.

The invention further provides a method for assessing the ability of anagent to modulate teneurin signalling, comprising the steps of: (a)contacting teneurin with at least one agent in the presence of acellular component; (b) detecting cleavage of said teneurin in thepresence or absence of said agents by use of a method of the invention;and (c) correlating the value obtained in step (b) with a value obtainedin the absence of said agent, and correlating a difference betweenvalues as an indication of the presence of an agent effective inmodulating teneurin signalling.

In another aspect, a method for assessing the ability of an agent tomodulate teneurin signalling is provided further comprising: (a)determining the presence and/or amount of cleaved teneurin product in acell; (b) exposing the cell expressing teneurin to the agent; (c)measuring the presence and/or amount of cleaved teneurin product in thepresence of the agent; and (d) comparing the determinations made in (a)and (c). Also provided is a method wherein step (b) is performed byperfusing the cell with the agent.

In another aspect, a method for assessing the ability of an agent tomodulate teneurin-mediated signalling is provided, comprising the stepsof: (a) measuring expression or activity of genes regulated by teneurinin cells; (b) exposing said cells to the agent; (c) measuring expressionor activity of genes regulated by teneurin in said cells in the presenceof the agent; and (d) correlating a modulated value in step (a) comparedto step (c) with the ability of the agent to regulate teneurinsignalling.

Also provided is a method of screening for compounds for the potentialto modulate cell proliferation or for compounds for the treatment ofneurodegenerative disease or disorder or for the treatment of cancer,which method comprises assessing the ability of said compounds tomodulate teneurin signalling by use of a method of the invention. Inanother embodiment, a method of screening is provided further comprisingthe subsequent step of isolation and/or manufacture and/or use in amethod of treatment, of an agent which tests positive.

In a further aspect, a composition detected by a method of screening ofthe invention is provided for the treatment or prophylactic treatment oftumourigenesis or cancer or for the treatment or prophylactic treatmentof neuropathology. The composition detected by a method of screening canbe used for the manufacture of a medicament for the treatment orprophylactic treatment of tumourigenesis or cancer or for themanufacture of a medicament for the treatment or prophylactic treatmentof neuropathology.

Also provided Is the use of a cleaved teneurin product for themanufacture of a medicament for the treatment or prophylactic treatmentof tumourigenesis or cancer or neuropathology.

Further provided is a composition comprising a cleaved teneurin productand a cellular target of the cleaved teneurin product, such as PML, Zic,p53, myc or ponsin.

DETAILED DESCRIPTION OF THE INVENTION

Intracellular pathways are known to be regulated by a cascade ofcomponents that undergo modulation in a temporally and spatiallycharacteristic manner. Several disease states can be attributed toaltered activity of individual signalling components (e.g., proteinkinases, protein phosphatases and transcription factors). Thesecomponents therefore render themselves as attractive targets fortherapeutic intervention, or as targets for agents that seek to obtain abiological effect by modulating a signalling pathway. Typically,modulation of a signalling pathway will alter the response of a cell toa particular stimulus in a variety of ways. Although much work has beencarried out in attempting to delineate the involvement of proteins insignalling pathways, prior to the present invention no hypothesis wasput forward linking teneurin to components of a signalling pathway.

The present inventors have demonstrated for the first time that teneurinis involved in intracellular signalling by its cytoplasmic domaininteracting with cellular targets that influence gene transcription andare involved in cerebellar development, neuronal differentiation,apoptosis, cell proliferation and senescence. Modulation of teneurinsignalling to these targets may therefore have application inneuropsychiatric, neurodevelopmental, and neurodegenerative diseases andother CNS diseases or conditions, as well as for the understanding ofneuronal differentiation, cell proliferation and oncogenesis.

The present inventors have found that teneurin is involved inintracellular signalling and exhibits transcriptional activity throughits cytoplasmic domain. The cytoplasmic domain of teneurin-2 can bereleased from the cell membrane and translocates to the cell nucleuswhere it is able to influence the transcription activity of varioustarget genes including zic-1, a vertebrate homologue of the Drosophilaopa. Teneurin signalling may be of importance in many neurodevelopmentaland neurodegenerative diseases and disorders, such as schizophrenia,Alzheimer's, Parkinsons disease, amyloid angiopathies, and otherdevelopmental disorders such as spina bifida and mental retardation. Inaddition teneurin Is thought to be important in adhesion, signalling,regulating cell survival, proliferation and differentiation and teneurinsignalling may have applications in cell proliferation, such as incancer.

Zic1, is a zinc finger transcription factor homologous to the Drosophilapair-rule gene opa, a possible downstream target of one of theDrosophila teneurins.

The present inventors have investigated the role of teneurin inintracellular signalling and found intracellular teneurin targets. Usinglive Imaging of cells expressing labelled or tagged proteins, theinventors showed that transfection of cells with a construct expressingthe cytoplasmic domain of teneurin-2 resulted in the translocation ofthe cytoplasmic domain to the nucleus of the cells. Its expression wasconfined to discrete spots within the nucleus in contrast to thestaining pattern of teneurin comprising the transmembrane domain ofteneurin-2, which showed accumulation on the cell surface (Example 1).The nuclear localisation coincided with a very similar punctuate patternobtained by staining for PML (promyelocytic leukemia), a protein whichlocalizes to the PML-nuclear bodies and which controls apoptosis, cellproliferation, and scenescence. An identical staining pattern wasdetected after co-transfection of the cytoplasmic domain of teneurin-2with PML, suggesting an involvement of the cytoplasmic domain ofteneurin-2 in transcriptional regulation.

Furthermore, the present inventors have demonstrated thatco-transfection of the cytoplasmic domain of teneurin-2 with Zic1reduced the transcriptional activity of Zic which suggests a role ofteneurin in the control of cerebellar development by regulating neuronaldifferentiation and other neurological conditions.

In a yeast-two-hybrid screen the cytoplasmic domain of teneurin-1 wasfound to bind to ponsin, a p53 responsive gene also involved inapoptosis and scenescence. The present inventors confirmed this bindingby co-transfection and co-immunoprecipitation of the two proteins.

Microarray analysis was performed using Affymetrix HG-U133A GeneChips™to identify genes that are regulated by teneurin signalling. Among thegenes up- and down-regulated by teneurin-2, many were shown to play arole in the regulation of cell growth, differentiation and apoptosis. Agroup of genes down-regulated by teneurin-2 correlated with genes knownto be glutathionylated and involved in the regulation of the redoxstatus of cells. Many of the teneurin-2 up-regulated genes are targetsof p53, whereas many of the teneurin-2 down-regulated genes are targetsof myc.

Together these data indicate that teneurin plays a role In intracellularsignalling pathways which has not been known from previous studies. Ingeneral terms, the present invention relates to a cleaved teneurinproduct as a specific marker for teneurin-mediated signalling.

Accordingly, a method for detecting teneurin signalling is provided,which method comprises (a) determining the presence and/or amount of acleaved teneurin product associated with the signalling; and (b)correlating the presence and/or amount of the cleaved teneurin productwith teneurin signalling. Optionally, the method further comprisesproviding a teneurin or a fragment thereof comprising at least a portionof the N-terminal domain of teneurin and at least a portion of theC-terminal domain of teneurin, and a cellular component that cleavesteneurin.

By ‘signalling’ is meant a process regulated by a cascade of componentsthat undergoes modulation in a temporally and spatially characteristicmanner (Gomperts et al., (2002), Signal transduction, San Diego,Academic Press, 424 p.; or Krauss (2001), Biochemistry of SignalTransduction and Regulation, Wiley-VCH, 2nd edition, 528 p.). Activityof individual intracellular signalling components (e.g., proteinkinases, protein phosphatases, transcription factors) can be altered inseveral disease states. Teneurin signalling can be detected by analysingthe modulation (up- or down-regulation) of downstream targets ofteneurin, which play a role in the regulation of cell growth,differentiation and apoptosis.

The teneurin protein provided for the method of the present inventionmay be mammalian teneurin, preferably mouse or rat teneurin, morepreferably human teneurin. It may be chosen from teneurin-1, teneurin-2,teneurin-3 or teneurin-4 (described in Minet and Chiquet-Ehrismann(2000) Gene 257: 87-97).

By a ‘fragment’ of teneurin is meant a fragment comprising a proteasecleavage site and at least a portion of the exodomain of teneurin, atransmembrane domain, and at least a portion of the cytoplasmic domainof teneurin. The portion will typically be at least one, preferably atleast ten or more amino acid residues in length.

A ‘cellular component’ in the meaning of the present invention has aprotease activity and Is capable of cleaving teneurin to provide afragment capable of signalling and affecting teneurin targets. Theactivity may regulate intramembrane proteolysis (RIP), where at leasttwo cleavage steps by proteases lead to the separation of theintracellular part from the membrane. The first cleavage and splittingoff of the truncated extracellular or intraluminal parts areprerequisite for the second cleavage. The latter takes place within thetransmembrane domain. The resulting soluble cytoplasmic part issubsequently translocated to the nucleus where it participates intranscription (reviewed in Brown et al. (2000) Cell 100, 391-98). Thecellular component of the present invention may thus work in conjunctionwith another protease activity, but comprises at least one proteasecapable of cleaving teneurin in or at the transmembrane domain resultingin a soluble cleaved teneurin product associated with teneurinsignalling. The protease may be selected from the group including,without limitation, site 1 proteases, site 2 proteases, or alpha-, beta-or gamma-secretase. These proteases have been shown to be involved inthe regulated intramembrane proteolysis (RIP) mediated nuclearsignalling (Ebinu and Yankner (2002) Neuron 34(4):499-502).

The cleaved teneurin product of the invention comprises the cytoplasmicdomain of teneurin or a fragment thereof and may be present, withoutlimitation, in tumour cells containing PML nuclear bodies or in neurons.The cytoplasmic domain of teneurin may be a polypeptide having the aminoacid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQID NO: 8, or a variant thereof such as a variant comprising an aminoacid deletion, addition (e.g. fusion proteins) or substitution relativeto the above mentioned amino acid sequences. The various nucleic acidsthat can encode these polypeptides therefore may differ because of thedegeneracy of the genetic code, in that most amino acids are encoded bymore than one triplet codon. The identity of such codons is well knownin this art, and this information can be used for the construction ofthe nucleic acids within the scope of the invention. Variants differfrom wild-type protein in having one or more amino acid substitutionsthat enhance, add, or diminish a biological activity of the wild-typeprotein.

By a ‘fragment of the cytoplasmic domain of teneurin’ is meant afragment of a polypeptide having a number of amino acid residues in therange of 5-400, more preferably 10-300, most preferably 20-200.

Exemplary functional equivalents or derivatives of the amino acidsequences shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ IDNO: 8 include molecules wherein the polypeptide of the invention iscovalently modified by substitution, chemical, enzymatic, or otherappropriate means with a moiety other than a naturally occurring aminoacid. Derivatives which retain common structural features can befragments of the protein of the Invention, in particular fragmentsmaintaining teneurin signalling acitivity.

Useful fragments may exhibit an epitope recognized by polyclonal ormonoclonal antibodies raised against the polypeptide having the aminoacid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQID NO: 8 for example. Particularly preferred fragments are thoseexhibiting a sequence capable of inducing or inhibiting teneurinsignalling.

The determination step in the method may be preceded by exposing thecell or the cell free system to ponasterone or to another putative orknown activating stimulus, including without limitation pharmacologicalagents, to induce expression of teneurin.

In some embodiments, the method for detecting teneurin signallingfurther comprises labelling teneurin or the cleaved teneurin productwith a detectable tag or label. In one aspect, the labelled teneurin isrecombinant, such as in the form of a fusion with another protein, forexample, tags for the targeted delivery or detection of the polypeptide(including fragments thereof). It is desirable that the tag or label iseasily detectable. In principle it may be inherently detectable (e.g. aprotein identifiable by a consonant antibody). Immunological methods ofdetection include, Western blot, immunohistochemical staining of cellsor tissue sections, and assays of cell culture, to quantitate directlythe expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared by conventional methodswell known in the art. Conveniently, the antibodies may be preparedagainst a native sequence of the tag or label.

In preferred embodiments the ‘label’ in the meaning of the presentinvention will include a specific detectable label for increased ease ofscoring and/or sensitivity. Most preferably the activity of the labelledprotein, or the protein itself, can be estimated photometrically(especially by fluorimetry or luminometry). This may be directly e.g.using for instance green fluorescent protein (GFP), yellow fluorescentprotein (YFP), insect luciferase or photobacterial luciferase.Alternatively, a radioactive or phosphorescent label may be used.

In another preferred embodiment, a ‘tag’ may be used wherein thedetection may be indirect e.g. whereby the signal gene causes a changewhich is detected by a colour indicator e.g. on staining. Other suitablesignal proteins (which have a readily detectable activity) are known inthe art e.g. β-galactosidase, which can generate a coloured product fromits substrate. The signal may utilise co-factors. The tag mayalternatively comprise a DNA binding domain which binds to nucleic acidcomprising regulatory sequences operably linked to a reporter gene. TheDNA binding domain may be, for example and without limitation, the GAL 4DNA binding domain. In another embodiment, the tag may further comprisean NFκB domain.

The method thus encompasses the detection of teneurin signalling bymeans of a cleaved teneurin product which is associated with thesignalling and a detectable tag or label in that the presence and\oramount of the cleaved teneurin product is increased when teneurinsignalling is activated. In general the tag or label under theconditions used in the method, will not be present (or detectablypresent) prior to contacting the cell or the cell free system withanother cellular component, a protease. The determination of thepresence and\or amount of the detectable tag or label attached to thecleaved teneurin product may be either qualitative or quantitative, andthe correlation may be based on comparison with the tag or label inunactivated systems (either directly, or based on historical orcontemporaneous comparison). For instance the amount of detectablecleaved teneurin product may be scored in each case and the scorescompared.

In order to produce suitable test systems including detectable cleavedteneurin product, it will generally be preferred to use nucleic acidencoding teneurin, which is either stably or transiently expressed. Anucleic acid encoding teneurin or its labelled or tagged variants maytherefore be introduced into a cell or a progenitor thereof to obtainexpression of the protein. Preferably, the teneurin is stably expressedin a cell. Alternatively, cells expressing teneurin can be used, such astumour cells, neurons or a progenitors thereof. In a preferredembodiment, cultured tumour cells are used. Alternatively, the teneurinmay be employed by in a cell free system and allowing detection with anantibody or using protein-protein interactions, for example.

Nucleic acids of, or for use in, the present invention (e.g. encodinglabelled teneurin) may be provided isolated from their naturalenvironment, in substantially pure or homogeneous form, or free orsubstantially free of other nucleic acids of the species of origin.Where used herein, the term “isolated” encompasses all of thesepossibilities. Nucleic acid according to the present invention may be inthe form of, or derived from, cDNA, RNA, genomic DNA and modifiednucleic acids or nucleic acid analogs. Thus the invention also relates,in a further aspect, to use of a nucleic acid molecule which comprises anucleotide sequence encoding teneurin described above linked to adetectable label, in the various methods of the Invention.

Nucleic acid sequences which encode a tagged polypeptide or peptidelinked to a label in accordance with the present invention can bereadily prepared by the skilled person using the information andreferences contained herein and techniques known in the art (forexample, see Sambrook, Fritsch and Maniatis, “Molecular Cloning, ALaboratory Manual”, Cold Spring Harbor Laboratory Press, 1989, andAusubel et al., Short Protocols in Molecular Biology, John Wiley andSons, 1992). These techniques include (i) the use of the polymerasechain reaction (PCR) to amplify samples of the relevant nucleic acid,e.g. from genomic sources, (ii) chemical synthesis, or (iii) preparationof cDNA sequences.

Thus in cell-based assays of the present invention, the labelled proteinof interest can be introduced by causing or allowing the expression in acell of an expression construct or vector. The construct may include anyother regulatory sequences or structural elements as would commonly beincluded in such a system, and as is described below. As well as thesignal sequence, the vector components will usually include, but are notlimited to, one or more of an origin of replication, one or more markergenes, an enhancer element, a promoter, and a transcription terminationsequence. Construction of suitable vectors containing one or more ofthese components employs standard ligation techniques which are known tothe skilled artisan.

Nucleic acid sequences which enable a vector to replicate in one or moreselected host cells are well known for a variety of bacteria, yeast, andviruses. For Example, various viral origins (SV40, polyoma, adenovirus,VSV or BPV) are useful for cloning vectors in mammalian cells.Particularly preferred is an expression vector comprising a nucleic acidas described herein. The vector may, for example, be in the form of aplasmid, cosmid, viral particle, phage, or any other suitable vector orconstruct which can be taken up by a cell and used to express thedetectable marker.

Expression vectors usually contain a promoter operably linked to theprotein-encoding nucleic acid sequence of interest, so as to direct mRNAsynthesis. Promoters recognized by a variety of potential host cells arewell known. “operably linked” means joined as part of the same nucleicacid molecule, suitably positioned and oriented for transcription to beinitiated from the promoter. DNA operably linked to a promoter is “undertranscriptional control” of the promoter. Transcription from vectors Inmammalian host cells is controlled, for example, by promoters obtainedfrom the genomes of viruses such as polyoma virus, fowlpox virus,adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus40 (SV40), from heterologous mammalian promoters, e.g. the actinpromoter or an immunoglobulin promoter, and from heat-shock promoters,provided such promoters are compatible with the host cell systems.

Expression vectors of the Invention may also contain one or moreselection genes. Typical selection genes encode proteins that (a) conferresistance to antibiotics or other toxins e.g. ampicillin, neomycin,methotrexate, or tetracycline, (b) complement auxotrophic deficiencies,or (c) supply critical nutrients not available from complex media, e.g.,the gene encoding D-alanine racemase for Bacilli.

The methos of the invention may therefore further include introducingthe nucleic acid into a host cell. The introduction, which may(particularly for in vitro introduction) be generally referred towithout limitation as “transformation”, may employ any availabletechnique. For eukaryotic cells, suitable techniques may include calciumphosphate transfection, DEAE-Dextran, electroporation, liposome-mediatedtransfection and transduction using retrovirus or other virus, e.g.vaccinia or, for insect cells, baculovirus. For example, the calciumphosphate precipitation method of Graham and van der Eb, Virology52:456-457 (1978) can be employed. General aspects of mammalian cellhost system transformations have been described in U.S. Pat. No.4,399,216. For various techniques for transforming mammalian cells, seeKeown et al., Methods in Enzymology, 185:527 537 (1990) and Mansour etal., Nature 336:348-352 (1988).

Host cells transfected or transformed with expression or cloning vectorsdescribed herein may be cultured in conventional nutrient media. Theculture conditions, such as media, temperature, pH and the like, can beselected by the skilled artisan without undue experimentation. Ingeneral, principles, protocols, and practical techniques for maximizingthe productivity of cell cultures can be found in “Mammalian CellBiotechnology: a Practical Approach”, M. Butler, ed. JRL Press, (1991)and Sambrook et al, supra.

There is no requirement that labelled teneurin of the present inventionhas to include the full-length sequence of the protein as it occurs innature. Variants may be used (e.g. which are derived from teneurin)which retain its activity. The term “derived” includes variants producedby modification of the authentic native sequence e.g. by introducingchanges into the full-length or truncated sequence, for examplesubstitutions, Insertions, and/or deletions. This may be achieved by anyappropriate technique, Including restriction of the coding sequence withan endonuclease followed by the Insertion of a selected base sequence(using linkers if required) and ligation. Also possible is PCR-mediatedmutagenesis using mutant primers. It may, for instance, be preferable toadd in or remove restriction sites in order to facilitate furthercloning.

Modified sequences according to the present invention may have asequence at least 60% identical to the sequence of teneurin. Typicallythere would be 80% or more, 90% or more 95% or more or 98% or moreidentity between the modified sequence and the authentic sequence. Theremay be up to five, for example up to ten or up to twenty or morenucleotide or amino acid deletions, insertions and/or substitutions madeto the full-length or part length sequence provided functionality is nottotally lost. Similarity or identity may be as defined and determined bythe TBLASTN program, of Altschul et al. (1990) J. Mol. Biol. 215:403-10, or BestFit, which is part of the Wisconsin Package, Version 8,Sep. 1994, (Genetics Computer Group, 575 Science Drive, Madison, Wis.,USA, Wisconsin 5371 1). Preferably sequence comparisons are made usingFASTA and FASTP (see Pearson & Lipman, 1988. Methods in Enzymology183:63-98). Parameters are preferably set, using the default matrix, asfollows: Gapopen (penalty for the first residue in a gap): -16 for DNA;Gapext (penalty for additional residues in a gap): -4 for DNA KTUP wordlength: 6 for DNA. Alternatively, homology in the context of nucleicacids can be judged by probing under appropriate stringency conditions.One common formula for calculating the stringency conditions required toachieve hybridization between nucleic acid molecules of a specifiedsequence homology is (Sambrook et al., 1989): T_(m)=81.5° C.+16.6 Log[Na+]+0.41 (% G+C)−0.63 (% formamide)−600/#bp in duplex.

The present inventors have demonstrated for the first time that thecytoplasmic domain of teneurin, the cleaved teneurin product of theinvention, contains transcriptional activity or acts as atranscriptional modulator which are characteristics of proteinsregulated by RIP (regulated intramembrane proteolysis). It could befound that the cytoplasmic domain of teneurin translocates to thenucleus and is capable of interacting with a cellular target. Thesetargets are found to include without limitation proteins belonging tothe ponsin/ArgBP2/venexin family (including without limitation ponsin,SH3P12, CAP, SORB1, ArgBP2, venexin, nArgBP2, FLAF2), preferably ponsin(see Example 3), nuclear targets such as the nuclear protein PML(promyelocytic leukemia, see Example 1), or transcription factors suchas zinc finger proteins which include without limitation Zic1-4, or Opr,preferably Zic1 (see Example 2), as well as other targets that areinvolved in neuronal differentiation or cell proliferation detected byexpression array analysis (Example 5, Table 1 and 2) which might includewithout limitation neuroserpin, protein kinase C (PKC) or any otherhitherto unknown target of teneurin.

In a further aspect, the present Invention provides compositions capableof enhancing or inhibiting expression or activity of the cellulartargets that regulate cell proliferation or neuronal differentiation.Therefore, depending on the function of its intracellular target, thecleaved teneurin product of the present invention is indirectly capableof enhancing or inhibiting transcriptional activation.

In a further aspect, the present invention provides a method fordetecting teneurin signalling wherein the cleaved teneurin productregulates expression or activity of a cellular target which itself is amodulator of cell proliferation or neuronal differentiation, which mayinclude, without limitation, PML, Zic, ponsin, p53-regulated genes,myc-regulated genes, or wherein the cleaved teneurin product targets tothe nucleus. The cellular target of the cleaved teneurin product may bedetected by expression array analysis.

Also provided is a method for detecting teneurin signalling wherein thepresence and/or amount of the detectable cleaved teneurin product iscorrelated to a particular disease. This method can be performed fordetermining whether a cleaved teneurin product is involved in cellproliferation or neuronal differentiation.

A ‘disease’ correlated to the cleaved teneurin product of the inventionmay mean any disease wherein cell proliferation or neuronaldifferentiation is affected, any cell pathology or neuropathologyincluding, without limitation, neurodevelopmental and neurodegenerativediseases and disorders such as schizophrenia, multiple sclerosis,Alzheimer's, Parkinsons disease, amyloid anglopathies, and otherdevelopmental disorders such as spina bifida or even mental retardation,as well as cancer. Targets of the cleaved teneurin product of theinvention have been shown to be involved in tumourigenesis (e.g. ponsinwhich is up-regulated by p53 expression, described in Kostic et al.(2000) Oncogene 19(35):3978-87), In neuronal differentiation andcerebellar development (reduced expression of zic results in spinabifida and holoprosencephaly, described in Aruga et al. (2002) Dev Biol.244(2):329-41), or in cell proliferation or apoptosis (PML, described inSalomoni and Pandolfi (2002) Cell 108 165-70).

In one embodiment, an in vitro method of diagnosis of a neuropathologyor cell pathology which affects teneurin-mediated signalling is providedwhich comprises performing the hereinabove described methods of theinvention on a cell which has been extracted from an animal which it Isdesired to diagnose.

The cleaved teneurin product disclosed herein may be used as adiagnostic. The expression of the cleaved teneurin product or theexpression or activity of target genes of the cleaved teneurin productmay be correlated to a particular disease state (which may include,without limitation, schizophrenia, Alzheimer's, Parkinsons disease,amyloid angiopathies, spina bifida, multiple sclerosis, mentalretardation, or cancer) and based on the diagnosis particular therapiescould be chosen. Thus the invention provides methods of diagnosis anduse of the materials disclosed herein in such methods, particularly inrespect of neuropathologies or cancer.

The invention further provides the use of a detectable cleaved teneurinproduct associated with teneurin signalling in an in vivo method ofdiagnosis of a neuropathology or cell pathology which affects teneurinsignalling is provided, which method comprises performing a method ofthe invention as hereinabove described.

For in vivo methods, host cells according to the present invention (i.e.including the detectable teneurin) may be comprised in a transgenicanimal, and the present invention further provides for a transgenicanimal, comprising cells which express a labelled teneurin, such as afusion protein of teneurin with a fluorescent protein, and also usesthereof. The transgenic organisms of the invention all include within aplurality of their cells a cloned recombinant or synthetic DNA sequencewhich encodes the labelled teneurin.

Since it is possible to produce transgenic organisms of the inventionutilizing a variety of labels and tags, a general description will begiven of the production of transgenic organisms by referring generallyto exogenous genetic material. This general description can be adaptedby those skilled in the art in order to incorporate the above-describedspecific DNA sequences into organisms and obtain expression of thosesequences utilizing the methods and materials described below. For moredetails regarding the production of transgenic organisms, andspecifically transgenic mice, refer to U.S. Pat. No. 4,873,191, issuedOct. 10, 1989 (incorporated herein by reference to disclose methodsproducing transgenic mice), and to the numerous scientific publicationsreferred to and cited therein.

The exogenous genetic material may be placed in either the male orfemale pronucleus of the zygote. More preferably, it is placed in themale pronucleus as soon as possible after the sperm enters the egg. Inother words, right after the formation of the male pronucleus when thepronuclei are clearly defined and are well separated, each being locatednear the zygote membrane. The male pronucleus of a fertilized mouse eggis the preferred site for addition of the exogenous genetic material ofthe present invention.

It is most preferred that the exogenous genetic material be added to themale DNA complement of the zygote prior to its being processed by theovum nucleus or the zygote female pronucleus. It is thought that theovum nucleus or female pronucleus release molecules which affect themale DNA complement, perhaps by replacing the protamines of the male DNAwith histones, thereby facilitating the combination of the female andmale DNA complements to form the diploid zygote.

Thus, it is preferred that the exogenous genetic material be added tothe male complement of DNA or any other complement of DNA prior to itsbeing affected by the female pronucleus. For example, the exogenousgenetic material is added to the early male pronucleus, as soon aspossible after the formation of the male pronucleus, which is when themale and female pronuclei are well separated and both are located closeto the cell membrane. Alternatively, the exogenous genetic materialcould be added to the nucleus of the sperm after it has been induced toundergo decondensation. Sperm containing the exogenous genetic materialcould then be added to the ovum or the decondensed sperm could be addedto the ovum with the exogenous genetic material being added as soon aspossible thereafter.

For the purposes of this invention a zygote is essentially the formationof a diploid cell which is capable of developing into a completeorganism. Generally, the zygote will be comprised of an egg containing anucleus formed, either naturally or artificially, by the fusion of twohaploid nuclei from a gamete or gametes. Thus, the gamete nuclei must beones which are naturally compatible, I.e., ones which result in a viablezygote capable of undergoing differentiation and developing into afunctioning organism. Generally, a euploid zygote is preferred. If ananeuploid zygote is obtained, then the number of chromosomes should notvary by more than one with respect to the euploid number of the organismfrom which either gamete originated.

In addition to similar biological considerations, physical ones alsogovern the amount of exogenous genetic material which can be added tothe nucleus of the zygote or to the genetic material which forms a partof the zygote nucleus. If no genetic material is removed, then theamount of exogenous genetic material which can be added is limited bythe amount which will be absorbed without being physically disruptive.Generally, the volume of exogenous genetic material inserted will notexceed about 10 picoliters. The physical effects of addition must not beso great as to physically destroy the viability of the zygote. Thebiological limit of the number and variety of DNA sequences will varydepending upon the particular zygote and functions of the exogenousgenetic material and will be readily apparent to one skilled in the art,because the genetic material, including the exogenous genetic material,of the resulting zygote must be biologically capable of initiating andmaintaining the differentiation and development of the zygote into afunctional organism.

The number of copies of the DNA sequences which are added to the zygoteis dependent upon the total amount of exogenous genetic material addedand will be the amount which enables the genetic transformation tooccur. Theoretically only one copy is required; however, generally,numerous copies are utilized, for example, 1,000-20,000 copies of agene, in order to insure that one copy is functional. As regards thepresent invention, there is generally an advantage to having more thanone functioning copy of each of the inserted exogenous DNA sequences toenhance the phenotypic expression of the exogenous DNA sequences.

Any technique which allows for the addition of the exogenous geneticmaterial into nucleic genetic material can be utilized so long as it isnot destructive to the cell, nuclear membrane or other existing cellularor genetic structures. The exogenous genetic material is preferentiallyinserted into the nucleic genetic material by microinjection.Microinjection of cells and cellular structures is known and is used inthe art.

Thus the present invention provides methods in which cloned recombinantDNA sequences encoding appropriate membrane targeting sequences may beinjected Into fertilized mammalian eggs (preferably mouse eggs). Theinjected eggs are implanted in pseudo pregnant females and are grown toterm to provide transgenic mice whose cells express proteins related tothe pathology of the relevant disease. The injected sequences areconstructed having promoter sequences connected so as to express thedesired protein in specific tissues of the transgenic mammal (mostnotably in nerve tissue). Examples may include the prion specificpromoter of Lewis et al (2000) Nature Genetics 25, 402-405, or theneurospecific enolase promoter.

Non-human animals of the invention may be homozygous or heterozygous forthe fusion polypeptide. Mammalian animals include non-human primates,rodents, rabbits, sheep, cattle, goats, pigs. Rodents include mice,rats, and guinea pigs.

Transgenic non-human mammals of the invention may, inter alia, be usedfor experimental purposes in studying neuronal development or cellproliferation and the development of therapies designed to alleviate thesymptoms of disease in which improper teneurin signalling is implicated.By “experimental” it is meant permissible for use in animalexperimentation or testing purposes under prevailing legislationapplicable to the research facility where such experimentation occurs.

An important aspect of the present invention is the use of thedetectable teneurin or of the detectable cleaved teneurin productdisclosed herein to screen for modulators affecting teneurin signalling,for example modulators (e.g. agonists or antagonists) affectingtranscription of genes regulated by teneurin and therefore affectingcell proliferation, apoptosis, neuronal differentiation, epilepsy,neurodegeneration, multiple sclerosis, schizophrenia, Alzheimer's,Parkinsons disease, amyloid angiopathies, and other developmentaldisorders such as spina bifida or even mental retardation. This can beachieved in the light of the disclosure herein by looking for compoundsthat would enhance (for agonists) or supress (for antagonists)expression of the cleaved teneurin product (or cause activation orredistribution of other proteins that are regulated by teneurin), orthat would enhance or supress protease activity of proteases that cleaveteneurin. Thus, an indicator for the cleaved teneurin product is usefulfor screening for compounds that improve cancerous conditions or induceneuronal regeneration.

Accordingly, the present invention provides a method for screeningcompounds for the modulation of teneurin signalling comprising assayingfor a modification of cleaved teneurin product, optionally labelled, andtargeting to the nucleus of a cell, such a s a transcription factor.

Thus, the invention provides a method for assessing the ability of anagent to modulate teneurin signalling, comprising the steps of: (a)contacting teneurin with at least one agent in the presence of one ormore cellular components (capable of cleaving teneurin); (b) detectingcleavage of said teneurin in the presence or absence of said agent byuse of a method of the invention as hereinabove described; and (c)correlating the value obtained in step (b) with a value obtained in theabsence of said agent, and correlating a difference between values as anindication of the presence of an agent effective in modulating teneurinsignalling. An increase in cleaved teneurin product correlates with anagent that is an agonist of teneurin signalling or an agonist of aprotease involved in the cleavage process of teneurin and may thereforebe used as an anti-tumour agent. A decrease In cleaved teneurin productcorrelates with an agent that is an antagonist of teneurin signalling oran antagonist of a protease involved in the cleavage process of teneurinand may therefore be used as an agent inhibiting neuronal degeneration.

Thus the various methods described above may comprise the further stepsof contacting the cleaved teneurin product, or proteases cleavingteneurin, with one or more agents which it is desired to assess forability to modulate teneurin signalling, and comparing the signalling inthe presence or absence of said agents—the relative values may becorrelated with its activity as a modulator.

Therefore, a method for assessing the ability of an agent to modulateteneurin signalling is provided further comprising: (a) determining thepresence and/or amount of cleaved teneurin product in a cell; (b)exposing the cell expressing teneurin to the agent; (c) measuring thepresence and/or amount of cleaved teneurin product in the presence ofthe agent; and (d) comparing the determinations made in (a) and (c).Also provided is a method wherein step (b) is performed by perfusing thecell with the agent.

In another aspect, a method for assessing the ability of an agent tomodulate teneurin-mediated signalling is provided, comprising the stepsof: (a) measuring expression or activity of genes regulated by teneurinin cells; (b) exposing said cells to the agent; (c) measuring expressionor activity of genes regulated by teneurin in said cells in the presenceof the agent; and (d) correlating a modulated value in step (a) comparedto step (c) with the ability of the agent to regulate teneurinsignalling.

Also provided is a method of screening for agents for the potential tomodulate cell proliferation for the treatment of cancer or diabetes orfor agents for the treatment of neurodegenerative disease or disorder,which method comprises assessing the ability of said agents to modulateteneurin signalling by use of a method of the invention.

Where the method of Identifying modulators utilizes a cell-based system,it may further include the step of testing the viability of the cellsexpressing the labelled cleaved teneurin product e.g. by use of alactate dehydrogenase assay kit (Sigma). This step may provide anindication of any interference by the test agent of vital cellularfunctions. Essentially, methods of the present invention may be employedanalogously to high throughput screens such as those well known in theart—see e.g. WO 200016231 (Navicyte); WO 200014540 (Tibotec); DE19840545 (Jerini Biotools); WO 200012755 (Higher Council for ScientificResearch); WO 200012705 (Pausch M H; Wess J); WO 200011216(Bristol-Myers Squibb); U.S. Pat. No. 6,027,873 (Genencor Intl.); DE19835071 (Carl Zeiss; F Hoffman-La Roche); WO 200003805 (CombiChem); WO200002899 (Biocept); WO 200002045 (Euroscreen); U.S. Pat. No. 6,007,690(Aclara Biosciences).

Agents which are tested may be any which it is desired to assess for therelevant signalling.

The methods can serve either as primary screens, in order to identifynew inhibitors/modulators, or as secondary screens in order to studyknown inhibitors/modulators in further detail.

‘Agents’ may be natural or synthetic chemical compounds. Relativelysmall chemical compounds, preferably which are capable of crossing theblood-brain barrier, may be preferred. Inhibition of the proteasesinvolved in the separation of the intracellular part of teneurin fromthe membrane part can block signalling and therefore inhibit or activatetranscription. Specific protease inhibitors could therefore beparticularly useful as potential agents for inhibting teneurinsignalling and could be used as potent tools to prevent generation ofsignalling products or as specific targets for cancer therapy.

The skilled person will appreciate that the amount of test substance orcompound which is added in a screening assay according to this aspect ofthe invention will normally be determined by trial and error dependingupon the type of compound used. It may be selected to be a level whichcould realistically be used in therapeutic context i.e. would benon-lethal to a patient. Typically, from about 0.01 to 100 nMconcentrations of putative modulator compound may be used, for examplefrom 0.1 to 10 nM.

In a further aspect, a composition detected by a method of screening ofthe invention is provided for the treatment or prophylactic treatment oftumourigenesis or cancer or for the treatment or prophylactic treatmentof neuropathology. The composition detected by a method of screening canbe used for the manufacture of a medicament for the treatment orprophylactic treatment of tumourigenesis or cancer or even diabetes, orfor the manufacture of a medicament for the treatment or prophylactictreatment of neuropathology or the inhibition of neuronal degeneration.

Performance of a screening assay method according to the various aspectsabove may be followed by isolation and/or manufacture and/or use of acompound, substance or molecule which tests positive for ability tointerfere with or modulate the neuronal differentiation, neuronaldevelopment or cell proliferation.

The compounds thus identified may be formulated into compositions foruse in the diagnosis, prognosis or therapeutic treatment. Thus, thepresent invention also extends, in further aspects, to pharmaceuticalformulations comprising one or more inhibitory or modulatory compound asobtainable by a screening method as provided herein.

A compound which has been identified as described above, may bemanufactured and/or may be used in the preparation, i.e. the manufactureor formulation, of a composition such as a medicament, pharmaceuticalcomposition or drug. These may be administered to individuals.

Not only the use of compounds which have been identified by a method ofscreening of the invention, but also the use of a cleaved teneurinproduct is provided for the manufacture of a medicament for thetreatment or prophylactic treatment of tumourigenesis or cancer orneuropathology.

Whether the compound which has been identified as described above is apolypeptide, antibody, peptide, nucleic acid molecule, small molecule,mimetic or other pharmaceutically-useful compound according to thepresent invention, or a cleaved teneurin product that is to be given toan individual, administration is preferably in a “prophylacticallyeffective amount” or a “therapeutically effective amount” (as the casemay be, although prophylaxis may be considered therapy), this beingsufficient to show benefit to the individual. The actual amountadministered, and rate and time-course of administration, will depend onthe nature and severity of what is being treated. Prescription oftreatment, e.g. decisions on dosage etc, is within the responsibility ofgeneral practitioners and other medical doctors.

Pharmaceutical compositions according to the present invention, and foruse in accordance with the present invention, may include, in additionto active ingredient, a pharmaceutically acceptable excipient, carrier,buffer, stabiliser or other materials well known to those skilled in theart. Such materials should be non-toxic and should not interfere withthe efficacy of the active ingredient. The precise nature of the carrieror other material will depend on the route of administration, which maybe oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may include a solid carriersuch as gelatin or an adjuvant. Liquid pharmaceutical compositionsgenerally include a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil. Physiological salinesolution, dextrose or other saccharide solution or glycols such asethylene glycol, propylene glycol or polyethylene glycol may beincluded.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, Isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

Examples of techniques and protocols mentioned above can be found inRemington's Pharmaceutical Sciences, 16^(th) edition, Osol, A. (ed),1980.

The agent may be administered in a localised manner to the brain orother desired site, e.g. tumour tissue, or it may be deliveredsystemically in a manner such that it targets the brain or other cells.

Further provided is a composition comprising a cleaved teneurin productand a cellular target of the cleaved teneurin product wherein saidtarget might be without limitation PML, Zic, or ponsin, or any targetidentified by expression array analysis (Table 1 and 2). The combinationof the cleaved teneurin product and its cellular target may be used as apharmaceutical or for the manufacture of a medicament for the treatmentor prophylactic treatment of tumourigenesis or cancer orneurodevelopmental or neuroregenerative disease. The production oradministration of such a pharmaceutical for use in the diagnosis,prognosis or therapeutic treatment will be performed as hereinabovedescribed.

EXAMPLES

The examples are described for the purposes of illustration and are notintended to limit the scope of the invention. Other embodiments of theinvention will occur to those skilled in the art in the light of these.

Methods of molecular genetics, protein and peptide biochemistry andimmunology referred to but not explicitly described in this disclosureand examples are reported in the scientific literature and are wellknown to those skilled in the art. For example, standard methods ingenetic engineering are carried out essentially as described in Sambrooket al., Molecular Cloning: A laboratory manual, 3rd Ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor N.Y., 2001 andantibody-related techniques according to: Using antibodies: a laboratorymanual/Ed Harlow, David Lane. Cold Spring Harbor, N.Y.: Cold SpringHarbor Laboratory Press, c1999. xiv, 495 p.)

Antibodies and DNA Constructs

The following antibodies and constructs are easily prepared by routinemethods and/or are available commercially: anti-teneurin-2 (Rubin et al.(1999) Dev Biol 216: 195-209), anti-VSV (affinity-purified peptideantibody described in Kreis (1986) Embo J 5,931-41), anti-FLAG (M2,Stratagene), anti-GAL4 (DGB RK5C1, Santa Cruz Biotechnology Inc.),anti-PML (PG-M3, Santa Cruz), anti-myc (c-Myc 9E10, Santa Cruz); anti-HA(1 2CA5, Roche); Alexa594- and Alexa488-conjugated goat-anti mouse andgoat anti-rabbit IgG (Molecular Probes), horseradish peroxidase coupledanti-mouse and anti-rabbit IgG (Soccochim). An antiserum was raisedagainst the N-terminal part of the cytoplasmic domain of teneurin-1(aminoacids 1-158; Minet et al. 1999) expressed as a His-tagged proteinin E. coli and purified by affinity chromatography on a Nickel column(Quiagen). The IgG fraction of this antiserum was purified over ProteinA Sepharose (Amersham) according to standard procedures.

Constructs: pFR-luc (luciferase reporter plasmid; Stratagene),pSV-β-Galactosidase (Promega), pCMV-AD, PCMV-BD and pBD-NFκB(Stratagene), p-CMX-PML and p-CMX-PML-RAR (described in Doucas et al.(1993) Proc Natl Acad Sci USA 90(20):9345-9), pEF-zic1 (described InAruga et al. (1996) J Biol Chem. 271(2):1043-7), pXP2-APOE1 89(luciferase reporter plasmid under the control of an apolipoprotein Epromoter; described in Salero et al., (2001) J Biol Chem. 276(3):1881-8)are used below.

Teneurin-2 Constructs

Seven different teneurin-2 constructs were used, these are namelyBDAD-CTEY, BDAD-CTE, BDAD-CT, BDAD-C, C, TE, TEY. They are namedaccording to the teneurin-2 protein domains contained within theircoding regions, namely CTEY (for Cytoplasmic, Transmembrane, EGF-likeand YD-repeats), containing the complete coding region of the long formof teneurin-2 including the cytoplasmic domain as well as the entirelong form of the extracellular domain (described in Tucker et al. (2001)Dev Dyn 220: 27-39, Accession No. AJ279031); TEY, containing the entirelong form of the extracellular domain; CTE, corresponding to a shortsplice variant of teneurin-2 containing the complete coding region ofthe short variant of teneurin-2 (Accession No. AJ245711) (described inRubin et al. (1999) Dev Biol 216: 195-209); or TE, the short splicevariant of teneurin-2 but without a cytoplasmic domain. Construct Crepresents the soluble cytoplasmic domain of teneurin-2. It encodes thefirst 372 amino acids of the teneurin-2 sequence as described in Rubinet al. 1999 (Rubin et al. (1999) Dev Biol 216: 195-209) followed by aVSV-G tag (an eleven amino acid sequence from residues 501 to 511 of thevesicular stomatitis virus (VSV) glycoprotein) for detection. In fourconstructs teneurin-2 was coupled to the GAL4 binding domain (BD) andthe NFκB activation domain (AD) generating BDAD-teneurin-2 fusionproteins. These constructs were cloned by multiple PCR. The product ofthe PCRs comprised bases 675-1118 of pCMV-BD coding for the GAL4 bindingdomain (BD), bases 703-1267 of pCMV-AD coding for the (AD) and bases1-630 of teneurin-2 coding for the first 210 aminoacids of thecytoplasmic domain of teneurin-2 until the BIpI site. These fragmentswere connected by the method of SOE (gene splicing by overlap extension,described in Horton R. M. (1995) Mol. Biotechnol., 3(2); 93-99) and theresulting construct was then cloned into the BamHI/Blpl site of thepreexisting pcDNA3/Neo vectors (Invitrogen) containing teneurin-2constructs of different lengths as described above.

Transient Transfections

HT1 080 human fibrosarcoma (ATCC CRL-12012) and COS-7 cells(SV40-transformed kidney fibroblasts from the African green monkey, ATCCCRL-1651) were routinely maintained In DMEM medium (Dulbecco's ModifiedEagles Medium) supplemented with 10% FCS (fetal calf serum). Fortransient transfections the cells were seeded in 6-well plates or 4-wellstaining dishes (Greiner). Twelve hours later they were transfected withthe expression vectors containing the constructs (1 μg of each vector)described above and in Example 2 (Zic), Example 4 and Example 5 by usingFUGENE-6 (3 μl, 6 μl or 9 μl for one, two to three, or four differentplasmids, respectively). The cells were treated with the followingsubstances at least five hours after transfection: ALLN (25 μg/ml;N-acetyl-leu-leu-norleu-AL; Sigma); tunicamycin (2 μg/ml; Sigma) orlactacystin (10 μM; e.g. Sigma) for four or eight hours prior toharvesting. Twenty-four hours after transfection the cells were rinsedin PBS (Phosphate-buffered saline) and processed for either measuringluciferase and β-galactosidase activities, western blotting orimmunofluorescence, essentially as described below.

Stable Cell Lines

Construct C was subcloned into the ecdyson-inducible expression vectorpIND (Invitrogen) and transfected into EcR 293 cells according to themanufacturer's manual (Invitrogen) (EcR cell lines have been stablytransformed with the regulatory vector, pVgRXR, for use in theEcdysone-lnducible Mammalian Expression System. The cell lines expressthe ecdysone receptor, which regulates muristerone-dependent inductionfrom pIND and pIND(SP1). Clones were tested for the inducible expressionof construct C by the addition of increasing concentrations ofPonasterone (1-10 μg/ml; Invitrogen) by immunoblotting using anti-VSVantibodies.

Luciferase and β-Galactosidase Assays

The cells were lysed by adding reporter lysis buffer (Promega). Serialdilutions of the lysed cell suspension were then pipetted intoMicrolite™ luciferase plates (Dynex Technologies) and the luciferaseactivity was measured in a Microlumat (LB96P, EG+G Berthold) byautomatic injection of luciferin substrate solution (2 mM luciferin, 100mM ATP in 250 mM glycin pH 7.8, 150 mM MgSO₄). All luciferase activitieswere normalised with respect to the transfection efficiency byco-transfecting a β-galactosidase vector. To determine β-galactosidaseactivity the diluted cell suspensions were incubated with the substratesolution (4.5 mM 2-nitrophenyl-β-D-galactopyranoside in 0.2 MNa-phosphate, 2 mM MgCl₂, 0.1 mM β-mercaptoethanol) for 30 min at 37° C.To stop the enzymatic reaction, three times the sample volume of 1 MNa₂CO₃ was added and the OD was measured at 405 nm in a microplatereader (BioRad).

Western Blotting

Teneurin-2 constructs C, CT and CTE were extracted by adding SDS samplebuffer containing 20% β-mercaptoethanol directly to the cells (100μl/3.5 cm plate). Extraction of the nuclear constructs BDAD and BDAD-Cwas achieved by performing nuclear fractionation. The transfected cellswere harvested by scraping off the cell layer in 200p1 lysis buffer (10mM HEPES pH 7.5, 0.5% triton X-1 00, 300 mM sucrose, 100 mM NaCl, 2 mMMgCl₂, protease inhibitors (Complete™, Roche Diagnostics) on ice andsubsequent centrifugation for 10 min at 2000 rpm in an Eppendorfcentrifuge. The resulting pellet was resuspended in lysis buffer andcentrifuged again. The final pellet was then dissolved in SDS samplebuffer containing 20% β-mercaptoethanol, 6 M urea and proteaseinhibitors (Complete™). Before loading on an 8% SDS-PAGE gel (sodiumdodecyl sulfate-polyacrylamide gel electrophoresis), DTT was added to afinal concentration of 10 mM.

The transmembrane constructs BDAD-CTE and BDAD-CTEY were extracted fromthe cells by the following procedure. The cells were extracted on ice by200 μl of a hypotonic buffer (2 mM Na-phosphate buffer pH 7.5, 20 mMKCl, 1 mM β-mercaptoethanol), scraped off and centrifuged for 10 min at8000 rpm at 4° C. in an Eppendorf centrifuge. The resulting pellet wasreconstituted in 100 μl of detergent buffer (50 mM Tris pH 8, 1% NP40,150 mM NaCl, 5 mM EDTA, 6 M urea, protease inhibitors (Complete™),incubated for 20 min at 37° C. and centrifuged for 10 min at 13'000 rpm.An equal volume of SDS sample buffer containing 20% β-mercaptoethanol, 6M urea and protease inhibitors (Complete™) was added to the supernatantand incubated for 1 hour at 52° C. After DTT was added (10 mM), thesamples were loaded on a 6% SDS-PAGE gel.

The gels were transferred to PVDF (polyvinylidene fluoride) membranes.The proteins were detected by anti-GAL4 antibody (BDAD and BDAD-C) or byanti-teneurin-2 serum (BDAD-CTE and BDAD-CTEY), horseradish peroxidasecoupled secondary antibodies and ECL SuperSignal® (Pierce).

Immunofluorescence

The cells grown on 4-well staining dishes (Greiner) were fixed with 4%PFA for 30 min at room temperature, and permeabilised with 0.1% TritonX-100 for 5 min. Incubation with primary antibodies was performed for 60min and that with secondary antibodies for 30 min both at roomtemperature whereas the cells were washed in PBS after each incubation.Finally the specimens were mounted in Moviol (Hoechst, FrankfurtGermany) and examined and photographed using a Zeiss Axiophot microscope(Carl Zeiss Ud.) connected to a 3CCD camera (Sony).

Microarray Analysis

Microarray analysis was performed using Affymetrix HG-U133A GeneChips™(Affymetrix, Santa Clara, USA). 10 μg of total RNA (isolated fromEcR-293 cells (Invitrogen)) was reverse transcribed using theSuperScript Choice system for cDNA synthesis (Life Technologies)according to the protocol recommended by Affymetrix (GeneChip ExpressionAnalysis: Technical Manual (2001) p. 2.1.14-2.1.16). The oligonucleotideused for priming was 5′-ggccagtgaattgtaatacgactcactatagggaggcgg-(t)₂₄-3′(SEQ ID NO: 9). Double-stranded cDNA was cleaned by phenol:chloroformextraction and the aqueous phase removed by centrifugation throughPhase-lock Gel (Eppendorf). In vitro transcription was performed on 1 μgof cDNA using the Enzo BioArray High Yield RNA transcript labelling kit(Enzo Diagnostics, USA) following the manufacturer's protocol. The cRNAwas cleaned using RNAeasy clean-up columns (Qiagen). To improve therecovery from the columns the elution water was spun into the matrix at27 g and then left for one minute prior to the standard 8000 gcentrifugation recommended by Qiagen. This low speed wetting step gavenearly double the yield of eluted RNA. The cRNA was fragmented byheating in 1× fragmentation buffer (40 mM Tris-acetate pH 8.1, 100 mMKOAc, 30 mM MgOAc). 10 μg of fragmented cRNA were hybridised to aHG-U133A GeneChip (Affymetrix) using their standard procedure (45° C.,16 hours). Washing and staining was performed in a Fluidics Station 400(Affymetrix) using the protocol EukGE-WS2v4 and scanned in an AffymetrixGeneChip scanner. Chip analysis was performed using the AffymetrixMicroarray Suite v5 (target intensity 500 used for chip scaling) andGeneSpring 4.2.1 (Silicon Genetics). Changes In gene expression wereassessed by looking for concordant changes between replicates using asigned Wilcoxon rank test (as recommended by Affymetrix). The “change”p-value threshold was <0.003 for increase and >0.997 for decrease. Afterconcordance analysis these values become <9×10⁻⁶ and >0.999991respectively. Any gene whose detection p-value was >0.05 in allexperimental conditions was discarded from the analysis as beingunreliable data.

Example 1 Localisation of the Teneurin-2 Cytoplasmic Domain to theNucleus and Its Co-Locallization with PML

This Example demonstrates that teneurin co-localizes with PML(promyelocytic leukaemia protein) in nuclear bodies. It was found thatthe cytoplasmic domain of teneurin-2 (referred to as construct C) istranslocated to the nucleus if transfected into human HT1080fibrosarcoma cells. Transfection of HT1080 cells with construct C led toaccumulation of the cytoplasmic domain in the nucleus, where itspresence was confined to discrete spots within the nucleus. In contrast,the immunofluorescence staining pattern obtained with the transmembraneversion of teneurin-2 (construct TEY) showed accumulation of the proteinon the cell surface.

The nuclear localisation reflected a very similar punctuate patternobtained by staining for PML protein (reviewed in Seeler et al. (1999)Curr Opin Genet Dev. 9(3):362-7; or Salomoni and Pandolfi (2002) Cell108,165-70) and is thus very suggestive of nuclear bodies termed PODs(promyelocytic oncogenic domains) or PML bodies.Double-immunofluorescent stainings of C transfected cells showedsubstantial overlap of PML staining with that of the cytoplasmic domainof teneurin-2.

Since PML bodies are involved in a number of functions associated withtranscriptional control (reviewed in Zhong et al. (2000) Nat Cell Biol.2(5):E85-90), the colocalisation of teneurin-2 and PML in nuclear bodieswas further investigated by cotransfecting C with PML, or C with PML-RAR(PML—retinoic acid receptor). Transfection of PML into cells containingendogenous PML protein leads to a massive enlargement of the nuclearbodies (Doucas et al. (1996) Biochim Biophys Acta. 1288(3):M25-9),whereas transfection of PML-RAR results in the destruction of the PMLbody architecture (Mu et al. (1994) Mol Cell Biol. 14(10):6858-67). Thepresent inventors also observed these effects. An equal staining patternwas detected after cotransfection of PML and C, where teneurin-2C (i.e.the cytoplasmic domain) was found in the enlarged PML bodies.Furthermore destruction of the PML body architecture after transfectionwith PML-RAR also changed the staining pattern with co-transfected C,which was no longer accumulated in discrete spots in the nucleus butseemed to be expressed homogeneously throughout the cells. These resultssuggest the presence of teneurin-2 C within nuclear bodies thussupporting a putative transcriptional regulatory function of thecytoplasmic domain of teneurin-2.

Example 2 Teneurin-2 Inhibits Zic Transcriptional Regulatory Activity

In this Example, it was determined whether the zinc finger transcriptionfactor Zic, a vertebrate homologue of opa would influence, or beinfluenced by, the cytoplasmic domain of teneurin-2. When both proteins,the cytoplasmic domain of teneurin-2 and Zic, were expressed in HT1080cells by transient transfections, a marked down-regulation (overten-fold) of teneurin-2 C was observed compared to its usual expressionlevel. In contrast, co-transfection of the two constructs had no effecton the Zic level. This effect was specific for teneurin-2 since BDAD wasnot down-regulated by the presence of Zic in an analogous analysis. TheZic induced downregulation of teneurin-2 C was counteracted by theaddition of the proteasome inhibitor lactacystin. By immunofluorescencestaining of the transfected cells we observed that Zic-transfected cellsrevealed a relatively diffuse nuclear staining of Zic and in nucleicontaining high amounts of Zic protein, the punctuate staining ofteneurin-2 C was not seen.

In order to examine a potential effect of the teneurin-2 C on thetranscriptional activity of Zic, EcR 293 cells stable transfectants wereproduced. In these cells teneurin-2 C was only expressed upon additionof ponasterone, which induces expression of the cytoplasmic domain ofteneurin-2. These cells were transiently transfected with Zic and theApoE-luciferase reporter construct known to be activated by Zic. Thepresence of Zic led to a dramatic increase in luciferase acitivity.After the induction of teneurin-2 C by ponasterone a marked reduction inthe expression level of the reporter gene was observed. These resultssuggested that the cytoplasmic domain of teneurin-2 has an inhibitoryeffect on the transcriptional activity of Zic and this effect is morepronounced in the presence of ALLN which was shown to stabilizeteneurin-2 C.

Example 3 Functional Interaction of Teneurin-1 with Ponsin

Yeast two hybrid screens using the cytoplasmic domains of teneurin-1 and-2 as bait were carried out to determine if teneurins interact withother proteins. The screen was performed with the DupLEX-A™ system(OriGene Technolgies, Inc.) according to their User's Manual (Version2.9 8/98). First, the entire cytoplasmic domains (aminoacids 1-300 ofteneurin-1; Minet et al. 1999 and aminoacids 1-376 of teneurin-2; Rubinet al. 1999) were used. However, both of these constructs resulted inself-activation of the LexA-dependent target genes and thus could not beused to search for interaction partners. We next split each of thecytoplasmic domains in two parts Ten-1 a, Ten-1 b and Ten-2a, Ten-2b,respectively (Ten-1a represents aminoacids 1-155 and Ten1b 156-300 ofteneurin-1 and Ten-2a represents aminoacids 1-168 and Ten2b 169-376 ofteneurin-2). Both, Ten-1a and Ten-2a were still self-activating theLeXA-dependent genes, implying a possible function for teneurins intranscriptional control. Ten-1b and Ten-2b could be used to screen awhole mouse 19day embryo DupLex-A cDNA library (OriGene). The screenwith Ten-1b resulted in an interacting cDNA clone that encoded the thirdSH3 domain of a protein called ponsin (Mandai et al. 1999).

The binding of full length ponsin to the cytoplasmic domain ofteneurin-1 was confirmed by co-transfection and co-immunoprecipitationof a myc-tagged posing expression plasmid (ponsin-2 in pCMV5-Myc asdescribed in Mandai et al. 1999) and the full length cytoplasmic domainof teneurin-1 (aminoacids 1-300; Minet et al. 1999) in Cos-7 cells.Cultures of Cos-7 cells (3.5 cm dishes at 70% confluency) wereco-transfected with 1 μg of each DNA using fugene (Roche). 48 hourslater cell layers were washed with PBS and the cell layers frozen on dryice to break up cells, which were then scraped into 300 μl of PBSincluding proteinase inhibitors (Complete proteinase inhibitor cocktailtablets, Roche). This cell extract was centrifuged at 14'000 rpm. To thesupernatant 150 μl of RIPA buffer (150 mmM NaCl; 50 mM Tris-HCl pH 8.0;1% NP-40; 0.5% Deoxycholic acid; 0.1% SDS; 50 mM NaF; 0.5 mM Na₃VO₄) wasadded followed by immunoprecipitation with 1 μg of anti-teneurin-1 IgG.The cell lysate was incubated with the anti-Ten-1 antibody for 90minutes slowly rotating at 4° C. Then 30 μl of protein A Sepharose beads(4 Fast Flow; Amersham) washed in RIPA buffer were added. After 60minutes of rotation at 4° C. the beads were collected by centrifugationand washed three times with PBS. The beads were eluted by boiling in 50μl of SDS-PAGE sample buffer and analyzed by western blotting. Theeluate was run in two parallel lanes on a 10% SDS-PAGE and blotted toPVDF membranes. The immunoprecipitated teneurin-1 was detected with ananti-FLAG antibody and the co-precipitated ponsin by anti-myc followedby secondary antibodies and detection using a chemiluminescence kit(ECL, Amersham). Whereas immunoprecipitated teneurin-1 contained ponsin,precipitates with control IgG did not contain any detectable ponsin.

The interaction of teneurin-1 with ponsin was further confirmed byimmunohistochemistry of co-transfected cells. Transfection of ponsinalone results in a cytoplasmic and cytoskeleton-associated expressionpattern, whereas the soluble cytoplasmic domain of teneurin-1accumulates in the nucleus. Upon co-transfection of the cytoplasmicdomain of teneurin-1 and ponsin, ponsin translocates together withteneurin-1 into the nucleus. Thus teneurin-1 co-localizes with and bindsto ponsin, which means that they will influence each others function inthe regulation of cell adhesion, cytoskeleton assembly and possiblytranscription.

Example 4 Morphological Changes of Cells Expressing the CytoplasmicDomain of Teneurin-2

Cell extracts from COS-7 green monkey kidney fibroblasts were prepared24 hours after transfection with CTEY, TEY, CTE and TE constructs andanalyzed by SDS-PAGE and immunoblotting with anti-teneurin-2 serum. Eachconstruct resulted in the presence of a major band of roughly theexpected size of 280 kD, 240 kD, 120 kd and 80 kd, respectively. Theproteins without the cytoplasmic tails (TEY and TE) were about 40kdsmaller than the corresponding proteins with these domains present (CTEYand CTE). Immunostaining of the transfected cells withoutpermeabilization prior to antiserum incubation revealed the presence ofthe extracellular domains of teneurin-2 on the cell surface. Themorphology of the cells expressing the constructs including thecytoplasmic domains was very different from the ones without thecytoplasmic domain. Whereas CTEY and CTE induced prominent filopodia inthe transfected cells, TEY and TE was present in cells with smoothsurfaces. This implies an interaction of the teneurin-2 cytoplasmicdomain with cytoskeletal components.

Example 5 Homophilic Binding of Teneurin Induces Cleavage

To isolate stable cell lines expressing teneurin-2 constructs humanHT1080 fibrosarcoma cells were transfected and replated to allow clonalgrowth of the transfected cells. Several clones representing theconstructs were screened for recombinant protein expression byimmunofluorescence as well as by immunoblotting. Phaloidin-staining anddetection of the recombinantly expressed teneurln-2 proteins with theanti-teneurin-2 antibody by immunohistochemistry was done as follows:

24 to 72 hours after transfection the cells were rinsed once with CA²⁺-and Mg²⁺-free PBS (PBS CMF) and either fixed with 4% formaldehyde in PBSCMF for immunohistochemistry or scraped off the dish and solubilized inSDS-PAGE sample buffer for Western blot analysis. To permeabilize thefixed cells 0.2% Triton X-100 in PBS CMF was applied for 15 min.following this step the permeabilized cells, like the intact cells, wererinsed with PBS CMF and incubated in blocking buffer (3% BSA in PBS CMF)for 15 min. The primary antibodies (anti-teneurin-2, anti-VSV tag, aswell as the fluorescein-coupled secondary antibody (JacksonImmunoResearch Laboratories, Inc., West Grove, Pa.) were diluted in theblocking buffer. Along with the secondary antibody phalloidin coupled torhodamine was incubated (Sigma, Buchs, Switzerland). After each of theincubations, cells were rinsed In PBS CMF. For viewing, cells weremounted in Moviol (Calbiochem, La Jolla, Calif.). For Western blotanalyses samples were separated on a 10% SDS-PAGE, transferred onto anylon membrane, and treated as described by Hagios et al. (1996) J CellBiol. 134(6):1499-512.

Teneurin-2 containing cellular extracts for immunoblotting were preparedas follows: After washing the cells on 5 cm culture dishes with coldPBS, plates were frozen at minus 20° C. After thawing, they wereextracted for 30 min on ice with 600 μl of hypotonic buffer (20 mMKCl, 2mM Na-phosphate pH 7.0/1 mM β-mercaptoethanol). Cells were collected byscraping with a rubber policeman and transferred into Eppendorf tubes.After spinning for 10 minutes at maximum speed the pellet was dissolvedat 37° C. for 20 min in 60 μl detergent buffer (150 mM NCl/50 mMTrispH8/1% NP-40/6M urea, 5 mM EDTA) per plate. An equal volume of SDS-PAGEsample buffer (0.2M Tris-HCl pH6.8/4%SDS/17.4% glycerol/20%β-mercaptoethanol/6M urea) was added and the sample was incubated for 1hour at 52° C. The samples were separated on SDS-PAGE and transferred toPVDF membranes. The teneurins were detected by the anti-teneurinantiserum (Rubin et al. (1999) Dev Biol. 216(1):195-209) and the signalsrevealed using the ECL western blotting system (Amersham).

We were able to select several clones of the two constructs, TEY and TE,that lack the cytoplasmic domain. The clones expressing the long form ofthe extracellular domain (TEY) showed a much flatter morphology thaneither the parental cells (HT1080) or the cells expressing the shortteneurin extracellular domain (TE) as can be seen both on the phasecontrast pictures as well as after phalloidin staining of the actincytoskeleton. Despite their flat morphology the clones of TEY did notcontain a more pronounced actin cytoskeleton than the parental cells andall cells revealed mostly cortical actin staining.

In addition to their flat phenotype we noticed that the cell clones ofTEY grew in epithelial cell-like patches suggestive of increasedcell-cell adhesion, whereas the parental cells and clone TE grew asdispersed fibroblast-like cells. Therefore, we investigated whether ornot these clones showed increased cell-cell adhesion in an aggregationassay of cells In suspension. Cells were harvested by incubation with0.2% EDTA, pelleted by centrifugation and resuspended at 2.5×10⁵cells/ml of Leibovitz L15 medium (Gibco) containing 1% fetal calf serum(Gibco). Three milliliters of each cell suspension were added to 5 cmbacterial plates (Sterilin) and incubated at 37° C. on a rotary shakerat 80 rpm. Photographs were taken at 15 min intervals and cellaggregation was analyzed by counting single cells versus cells indouble, triple or higher number aggregates. The aggregation index wascalculated as N_(o)-N_(t)/N_(o), where N_(o) is the initial number ofparticles corresponding to the total number of cells, and N_(t) is thenumber of remaining particles at the incubation time point t. While theparental HT1080 cells as well as the cells containing the TE constructremained as single cells, all clones expressing the long teneurin-2extracellular domain (TEY) aggregated into clumps of cells. The presenceof the extracellular domain of teneurin-2 on these cells resulted intheir homophilic aggregation. Quantitative comparison of the cellaggregation showed that after 30 minutes of incubation all TEY celllines had already approached maximal aggregation values, whereas theHT1080 and TE cells did not aggregate even after 90 minutes ofincubation.

Further the effect of the expression of the teneurin-2 constructs onNb2a neuroblastoma cells was investigated. As reported in Rubin et al.(1999) Dev Biol. 216(1):1 95-209, expression of CTE constructs in Nb2acells led to the induction of filopodia and enlarged growth cones. Thiseffect was not seen after transfection of the TE construct lacking thecytoplasmic domain and the protein mainly localized to cell bodies andnot to neurites. This suggests that the cytoplasmic domain is requiredfor the translocation of the teneurin protein to neurites and growthcones. The transfection of the longer constructs CTEY and TEY resultedin quite different teneurin-2 expression patterns. Both of theseproteins were expressed on the cell surfaces and were heavily enrichedin cell-cell contact areas giving further support for the promotion ofhomophilic interactions between these neuronal cells. The CTEYexpression led in addition to the accumulation of actin to theseteneurin-rich cell-cell contacts as revealed by phalloidin staining,which was not the case for the TEY construct. This provides furthersupport for an interaction between teneurin-2 and the actin cytoskeletonthrough its cytoplasmic domain.

Example 6 Cleavage and Stabilization of the Cytoplasmic Domain

The nuclear localization of the cytoplasmic domain and itstranscriptional regulatory role would mean that the wild-typetransmembrane teneurin-2 would have to be specifically cleaved in or atthe plasma membrane possibly upon ligand binding. The cytoplasmic domainwould thereby be released and translocated to the nucleus. To establishthis process, fusion proteins of full length teneurin-2 or smallerfragments comprising the transmembrane domain, attached to a GAL4 DNAbinding domain (BD) and a NFκB activation domain (AD) were introducedInto HT-1080 and Cos-7 cells. Cleavage of the fusion protein andtranslocation of BDAD-C to the nucleus can be detected by binding of BDto specific GAL4 binding sequences on a co-transfected luciferasereporter plasmid, which results in the subsequent initiation ofluciferase gene expression through AD.

BDAD and BDAD-C serving as positive controls In this experiment weredetectable on a Western blot of nuclear extract by anti-GAL4 antibodies.In addition, BDAD-C accumulation in the nucleus was confirmed byimmunofluorescent staining of permeabilised cells. At the same timeBDAD-CTE and BDAD-CTEY could be identified as part of the (plasma)membrane by western blot of a membranous cell fraction and byimmunofluorescence of non-permeabilised cells.

For analysis of the luciferase activity induced by the teneurin-2 fusionconstructs, HT1080 cells were cotransfected with the respectiveBDAD-teneurin-2 constructs, the luciferase reporter plasmid as well as aβ-Galactosidase construct for normalisation of transfectionefficiencies. BDAD-CTE, BDAD-CT and BDAD-C led to an induction ofluciferase activity over the negative control (BD construct). However,BDAD-CTEY being the largest fusion protein did not lead to asignificantly enhanced activity. Cleavage of the shorter constructsmight be constitutive while cleavage of the full length construct mighthave to be specifically induced, for example by ligand binding.

As described in Example 5, teneurin-2 has been shown to bindhomophilically by its extracellular domain and may induce cleavage andtranslocation of the cytoplasmic domain of the BDAD-teneurin-2 fusionproteins. To test whether homophilic interaction of teneurin-2represents a signal for cleavage of its cytoplasmic domain, BDAD-CTEYand as control BDAD-CT, were transfected into HT-1 080 cell clones thatconstitutively express TEY or TE on their surface (described in Example5 hereinabove) and the luciferase activity produced from theco-transfected reporter plasmid analysed. The luciferase activitiesobtained were normalized to the transfection of the same constructs intowild type HT1080 cells. Luciferase activity obtained after transfectionof BDAD-CTEY into the TEY cells was approximately 6-fold higher thanafter transfection of BDAD-CT while in TE cells the activity was notaffected by the type of construct used for transfection. Thus, theC-terminal of the extracellular domain is required to induce thecleavage of the cytoplasmic domain of teneurin-2.

In summary, the activity of the luciferase reporter gene originates fromthe cleavage of the BDAD-teneurin-2 fusion proteins at (or in thevicinity of) the membrane because it could be specifically upregulatedby homophilic binding of full length but not shorter truncated versionsof teneurin-2 extracellular domains on the surface of the cell. BDADattached to full length teneurin-2 (BDAD-CTEY) led to a significantinduction of the luciferase gene only when processing was upregulated byhomophilic binding of the extracellular C-terminal part of teneurin-2.In addition, this activation was not observed with the truncatedteneurin-2 fusions described above. Nevertheless, the shortercounterparts seemed to be cleaved without stimulation. Although notwishing to be bound by theory, the large BDAD-CTEY construct isexpressed less efficiently than its shorter counterparts and thecleavage products might be down-regulated quickly in proteasomes. Theinduction of luciferase activity following transfection of BDAD-CTEYcould indeed be markedly upregulated by the addition of proteaseinhibitors such as ALLN and lactacystin. Similarly, CTE protein levelsalso increased as demonstrated by western blot. The addition of ALLN ledto the stabilisation of two particular cleavage products, one of whichcorresponded to the size of the cytoplasmic domain alone.

Example 7 Identification of Genes Regulated by Teneurin Signalling

The cDNA encoding the cytoplasmic domain of teneurin-2 containing aC-terminal VSV-tag was cloned into the vector plnd (Invitrogen). EcR-293cells (Invitrogen) containing the stably integrated plasmid pVgRXR(Invitrogen) were transfected with the plnd teneurin-2 plasmid encodingthe cytoplasmic domain. Stable clones were selected and tested for theexpression of the cytoplasmic domain of teneurin-2 after addition ofponasterone (Invitrogen). Cells of one positive clone termed K3 was usedfor the further analysis. Parental EcR-293 cells and K3 cells were grownto 70% confluency before 10 μM ponasterone was added to both cellcultures for 24 hours. Parallel cultures were kept without the additionof ponasterone. After 24 hours mRNA was isolated using Trizol reagent(Gibco) from four different cultures: K3−ponasterone, K3+ponasterone,ECR293−ponasterone and ECR293+ponasterone. This procedure was typicallyperformed in duplicates, resulting in 8 mRNA batches. Each of thesebatches of mRNA was used to make probes to hybridyze to Affymetrix chips(U133) containing oligonucleotides for the detection of 20'000 potentialtranscripts. Lists were derived that contained genes that weredifferentially expressed depending on the presence of ponasterone. Genesthat were common to both lists of the two indepently performedduplicates were investigated further and made up the entries of the fourlists K3 up, K3 down (genes up- or down-regulated by ponasterone in K3cells), EcR-293 up, EcR-293 down (genes up- or down-regulated byponasterone in EcR-293 cells. To exclude any genes that were affected byponasterone directly, any entries common to the lists of the ECR293 andK3 cells were discarded resulting in the final lists of genesup-regulated (Table 1) or down-regulated (Table 2) by the presence ofthe cytoplasmic domain of teneurin-2. Table 1 shows the genesup-regulated by the cytoplasmic domain and the ID number of therespective database assigned by Affymetrix. Table 2 shows the genesdown-regulated by the cytoplasmic domain and the ID number of therespective database assigned by Affymetrix. Among the genes up- anddown- regulated by teneurin-2 many are known to play a role in theregulation of cell growth, differentiation and apoptosis. A group ofgenes up-regulated by teneurin-2 correlates with genes known to betargets of p53 (Nr. 7, 23, 33, 51, 65 in Table 1). Another group ofdown-regulated genes corresponded to genes known to play a role inneuronal differentiation and synapse formation and function (Nr. 4, 5,11, 12, 27, 28, 29, 30, 34, 35, 40, 56, 90 of Table 1). A group of genesdown-regulated by teneurin-2 correlated with genes known to beglutathionylated and involved in the regulation of the redox status ofcells (Nr. 3, 4, 11, 44, 46, 69 of Table 2). Another group ofdown-regulated genes corresponded to genes known to be targets of myc(Nr. 1, 3, 4, 11, 14, 31, 69 of Table 2).

Example 8 Proliferation Assay

The effect of the expression of the cytoplasmic domain of teneurin-2 oncell proliferation was tested. K3 cells (EcR-293 cells (Invitrogen)containing the stably integrated plasmid pVgRXR (Invitrogen) and theplnd teneurin-2 plasmid encoding the cytoplasmic domain, described inExample 7) were cultured in the presence or absence of ponasterone,which induces expression of the cytoplasmic domain of teneurin-2.Typically, over a 5 day period, parallel cultures were fixed with 4%formaldehyde every day and cell numbers were determined by staining thecells with crystal violet (0.1% in H₂0). In the presence of ponasteroneand thus in the presence of the cytoplasmic domain of teneurin, the K3cells were retarded in their growth rate to about 50% of the untreatedcells. Proliferation of EcR-293 cells without transfected cytoplasmicdomain of teneurin-2 was not affected by the addition of ponasterone,proving that the growth retardation was specifically dependent on thepresence of the cytoplasmic domain of teneurin-2.

Example 9 The Cytoplasmic Domain of Teneurin-1 Can Be Detected in Nucleiof C. elegans Embryo Cells

Ten-1 is the C. elegans orthologue of the Drosophila pair-rule geneten-m. We describe here the gene structure of ten-1 encompassing twopromoters that control the expression of two different ten-1transcripts. This results in the expression of two type II transmembraneprotein variants differing In their cytoplasmic domains. The 5′ ends ofthe ten-1 cDNAs were determined by RT-PCR using splice leader 1 (SL1) asthe 5′ primer and a ten-1 specific oligonucleotide as the 3′ primer.cDNA fragments were prepared by RT-PCR using Superscript RNaseH(−)reverse transcriptase (Life Technologies). 5′ cDNA ends were amplifiedwith Expand HiFi polymerase (Roche) using SLi as a 5′ primer and thegene specific primer AGCACGTGTCGCTATCGTCG (SEQ ID NO: 10) using cDNAprepared from mixed stage worms. The RT-PCR reaction using mixed stagemRNA as template resulted in two products differing in size. Bysequencing these bands two different cDNA species could be identified.One of them corresponded with a minor difference at the 5′ end to thetranscription start of the predicted open reading frame R16F6.4 and theother one contained in addition the predicted open reading frame F28F5.1together with a newly discovered exon. Thus the ten-1 gene is under thecontrol of alternative promoters resulting in two different transcriptsthat encode two Ten-1 proteins differing in their N-terminal sequences.The long form contains N-terminal to a predicted transmembrane sequencea cytoplasmic domain of 218 aa whereas the short form has only 37 aathat are in common between both variants. The intracellular sequences donot contain any predicted domains but contain a consensus sequence fortyrosine phosphorylation. The long form harbors a proline-rich stretchat the N-terminus and a potential bipartite nuclear localization signal.The N-terminal cytoplasmic domains are followed by a transmembranedomain making Ten-1 a type II transmembrane protein.

For DNA constructions 4 Kb of the upstream promoter where amplified withthe primers CATTGGTCMTTGGCGCGCCCATTCGCAGACG (SEQ ID NO: 11) andATTAGGCGGTGGGGGTACCGCATTCG (SEQ ID NO: 12) and cloned into the Ascl/Kpnlsites of pPD117.28 (A. Fire). 3 Kb of the downstream promoter whereamplified with the primers GMTTCGCATGCAAATGTGMGCATG (SEQ ID NO: 13) andCCACCAGGTACCGGATCACCATTGTTC (SEQ ID NO: 14) and cloned into theSphl/Kpnl sites of pPD1 17.28 (A. Fire). DNA encoding the longintracellular domain was amplified with the primersCAGAGTGCGGCCGCCCGTGCGTTTCG (SEQ ID NO: 15) andGGCTAGGMTTCATTCCATTTGGATGG (SEQ ID NO: 16). DNA encoding the shortintracellular domain was amplified with the primersTTACAATTTTTCAGGCGGCCGCAAGTTGGC (SEQ ID NO: 17) andGGCTAGGAATTCATTCCATTTGGATGG (SEQ ID NO: 18).

For antibody production, anti-Ten-1 antibodies ware raised against theTen-1 specific peptides from the N-terminus of the long variant(MFQHRTTNAQGPPPNRPMPR) (SEQ ID NO: 19) and the common C-terminus(PAHQSGLLASVHSWKFRKSE) (SEQ ID NO: 20). The peptides where synthesizedand the rabbits immunized at Neosystem (Strasburg, France). All serawere affinity purified using the respective peptides coupled toCNBr-activated Sepharose 4B columns according to standard procedures.

Antibodies against the N-terminus of the long ten-1 variant (anti-N) andthe one against the C-terminus of the protein (anti-C) were used tostain early embryos of C. elegans. Anti-C stained all membranes whileanti-N stained in addition also the nuclei of the Ten-1 expressingcells. This was also prevalent in the cells lining the gut where anti-Cstained the cell membranes and anti-N stained the predominantly thenuclei. Although not wishing to be bound by theory, the endogenous Ten-1might normally be proteolytically processed and the translocation of thecytoplasmic domain could be a physiological process in Ten-1 signaling.The nuclear translocation of the long cytoplasmic domain of Ten-1 can beconfirmed by overexpression of both cytoplasmic domains as GFP fusionproteins under a heat shock promoter. Transient expression of theseconstructs revealed that the long cytoplasmic domain accumulated in thenuclei of the cells whereas the short form remained cytoplasmic.

Ten-1 was characterized as a novel transmembrane protein of C. elegansand is required for gametogenesis, early embryogenesis, and hypodermalcell migration. In later stages of development it is involved inneuronal migration and pathfinding, distal tip cell migration andestablishing of the somatic gonad. Furthermore, it is also required forpharynx and gut development as well as for proper defecation. Althoughnot wishing to be bound by theory, Ten-1 might act as a receptor formorphogenetic cues and it directly signals to the nucleus by proteolyticrelease of its cytoplasmic domain from the cell membrane and bytranslocation to the nucleus. TABLE 1 Locus Ref Seq Unigene Link(NM_)/Gene Nr. Affimetrix ID ID ID Bank ID Ref Seq ID + Annotation 1200620_at Hs.11441 9528 NM_004872.1 NM_004872 chromosome 1 open readingframe 8 2 200710_at Hs.82208 37 NM_000018.1 NM_000018 acyl-Coenzyme Adehydrogenase, very long chain precursor 3 200804_at Hs.74637 7009NM_003217.1 NM_003217 testis enhanced gene transcript (BAX inhibitor 1)4 200848_at Hs.4113 10768 AA479488 NM_006621 S- adenosylhomocysteinehydrolase- like 1 5 200849_s_at Hs.4113 10768 AA479488 NM_006621 S-adenosylhomocysteine hydrolase- like 1 6 201057_s_at Hs.7844 2804NM_004487.1 NM_004487 golgi autoantigen, golgin subfamily b, macrogolgin(with transmembrane signal), 7 201236_s_at Hs.75462 7832 NM_006763.1NM_006763 BTG family, member 2 8 201301_s_at Hs.77840 307 BC000182.1NM_001153 annexin IV 9 201302_at Hs.77840 307 NM_001153.2 NM_001153annexin IV 10 201494_at Hs.75693 5547 NM_005040.1 NM_005040prolylcarboxypeptidase (angiotensinase C) 11 201565_s_at Hs.180919 3398NM_002166.1 NM_002166 inhibitor of DNA binding 2, dominant negativehelix-loop-helix protein 12 201566_x_at Hs.180919 3398 D13891.1NM_002166 inhibitor of DNA binding 2, dominant negative helix-loop-helixprotein 13 201637_s_at Hs.82712 8087 NM_005087.1 NM_005087 fragile Xmental retardation-related protein 1 14 201663_s_at Hs.50758 10051NM_005496.1 NM_005496 SMC4 structural maintenance of chromosomes 4-like1 15 201813_s_at Hs.115740 9779 NM_014744.1 NM_014744 KIAA0210 geneproduct 16 201814_at Hs.115740 9779 NM_014744.1 NM_014744 KIAA0210 geneproduct 17 201876_at Hs.169857 5445 NM_000305.1 NM_000305 paraoxonase 218 201964_at Hs.154919 23064 N64643 NM_015046 KIAA0625 protein 19201965_s_at Hs.154919 23064 NM_015046.1 NM_015046 KIAA0625 protein 20202436_s_at Hs.154654 1545 NM_000104.2 NM_000104 cytochrome P450,subfamily I (dioxin-inducible), polypeptide 1 21 202437_s_at Hs.1546541545 NM_000104.2 NM_000104 cytochrome P450, subfamily I(dioxin-Inducible), polypeptide 1 22 203232_s_at Hs.74520 6310NM_000332.1 NM_000332 ataxin 1 23 203409_at Hs.77602 1643 NM_000107.1NM_000107 damage-specific DNA binding protein 2 (48 kD) 24 203411_s_atHs.77886 4000 NM_005572.1 NM_005572 lamin A/C 25 203560_at Hs.78619 8836NM_003878.1 NM_003878 gamma-glutamyl hydrolase (conjugase,folylpolygammaglutamyl hydrolase) precursor 26 203594_at Hs.27076 8634NM_003729.1 NM_003729 RTC domain containing 1 27 203789_s_at Hs.17192110512 NM_006379.1 NM_006379 sema domain, immunoglobulin domain (Ig),short basic domain, secreted, (semaphor 28 203810_at Hs.41693 11080BG252490 NM_007034 DnaJ (Hsp40) homolog, subfamily B, member 4 29203999_at Hs.154679 6857 NM_005639.1 NM_005639 synaptotagmin I 30205352_at Hs.78589 5274 NM_005025.1 NM_005025 protease inhibitor 12(neuroserpin) 31 205934_at Hs.153322 5334 NM_006226.1 NM_006226phospholipase C, epsilon 32 207071_s_at Hs.154721 48 NM_002197.1NM_002197 aconitase 1 33 208478_s_at Hs.159428 581 NM_004324.1 NM_004324BCL2-associated X protein, isoform beta NM_138761 BCL2-associated Xprotein, isoform alpha NM_38762 BCL2-associated X protein, isoform gammaNM_138763 BCL2-associated X protein, isoform delta NM_138764BCL2-associated X protein, isoform epsilon 34 208786_s_at Hs.12184981631 AF183417.1 NM_022818 microtubule-associated proteins 1A/1B lightchain 3 35 208890_s_at Hs.3989 23654 BC004542.1 NM_012401 plexin B2 36208933_s_at Hs.70333 51322 AI659005 NM_016628 WW domain-containingadapter with a coiled-coil region, isoform 1 NM_018604 NM_100264 WWdomain-containing adapter with a coiled-coil region, isoform 2 NM_100486WW domain-containing adapter with a coiled-coil region, isoform 3 37208943_s_at Hs.8146 7095 U93239.1 NM_003262 translocation protein 1 38209091_s_at Hs.136309 51100 AF263293.1 NM_016009 SH3-containing proteinSH3GLB1 39 209167_at Hs.5422 2824 AF016004.1 40 209238_at Hs.82240 6809BE966922 NM_004177 syntaxin 3A 41 209268_at Hs.6650 11311 AF165513.1NM_007258 NM_007259 vacuolar protein sorting 45A NM_007259 vacuolarprotein sorting 45A 42 209295_at Hs.51233 8795 AF016266.1 NM_003842tumor necrosis factor receptor superfamily, member 10b 43 209312_x_atHs.308026 3123 U65585.1 NM_002124 major histocompatibility complex,class II, DR beta 1 precursor 44 209707_at Hs.62187 10026 AF022913.1 45209849_s_at Hs.11393 5889 AF029669.1 NM_002876 RAD51 homolog C, isoform2 NM_058216 RAD51 homolog C, isoform 1 NM_058217 RAD51 homolog C,isoform 3 46 209993_at Hs.21330 5243 AF016535.1 NM_000927 ATP-bindingcassette, sub-family B (MDR/TAP), member 1 47 210038_at AL137145 48210039_s_at Hs.211593 5588 L01087.1 NM_006257 protein kinase C, theta 49211061_s_at Hs.172195 4247 BC006390.1 NM_002408 alpha-1,6-mannosyl-glycoprotein beta-1,2-N- acetylglucosaminyltransferase 50 211769_x_atHs.272168 10955 BC006088.1 NM_006811 tumor differentially expressed 1 51211833_s_at Hs.159428 581 U19599.1 NM_004324 BCL2-associated X protein,isoform beta NM_138761 BCL2-associated X protein, isoform alphaNM_138762 BCL2-associated X protein, isoform gamma NM_138763BCL2-associated X protein, isoform delta NM_138764 BCL2-associated Xprotein, isoform epsilon 52 212120_at Hs.250697 23433 BF348067 NM_012249ras-like protein 53 212266_s_at Hs.166975 6430 AW084582 NM_006925splicing factor, arginine/serine-rich 5 54 212359_s_at Hs.65135 23053W89120 55 212408_at Hs.234265 26092 AK023204.1 56 212820_at Hs.1326423312 AB020663.1 NM_015263 rabconnectin-3 57 212998_x_at Hs.73931 3119AI583173 NM_002123 major histocompatibility complex, class II, DQ beta 1precursor 58 213056_at Hs.96427 23150 AU145019 59 213258_at Hs.288582BF511231 60 213400_s_at Hs.76536 6907 AV753028 NM_005647 transducinbeta-like 1X 61 213624_at Hs.42945 10924 AA873600 62 214098_at Hs.2155423285 AB029030.1 63 214449_s_at Hs.250697 23433 NM_012249.1 NM_012249ras-like protein 64 215719_x_at X83493.1 65 215785_s_at Hs.258503 26999AL161999.1 66 216231_s_at Hs.75415 567 AW188940 NM_004048beta-2-microglobulin 67 216860_s_at Hs.339699 10220 AF028333.1 NM_005811growth differentiation factor 11 68 217127_at AL354872 69 217731_s_atHs.239625 9445 NM_021999.1 NM_021999 integral membrane protein 2B 70217858_s_at Hs.172788 51566 NM_016607.1 NM_016607 ALEX3 protein 71218007_s_at Hs.108957 51065 NM_015920.1 NM_015920 ribosomal protein S27-like protein 72 218084_x_at Hs.333418 53827 NM_014164.2 NM_014164 FXYDdomain- containing ion transport regulator 5 73 218107_at Hs.28906980232 NM_025160.1 NM_025160 hypothetical protein FLJ21016 74 218113_atHs.160417 23670 NM_013390.1 NM_013390 transmembrane protein 2 75218132_s_at Hs.15580 79042 NM_024075.1 NM_024075 LENG5 protein 76218248_at Hs.19525 63901 NM_022074.1 NM_022074 hypothetical proteinFLJ22794 77 218341_at Hs.72531 79717 NM_024664.1 NM_024664 hypotheticalprotein FLJ11838 78 218603_at Hs.6679 51696 NM_016217.1 NM_016217 hHDCfor homolog of Drosophila headcase 79 218634_at Hs.268557 23612NM_012396.1 NM_012396 pleckstrin homology-like domain, family A, member3 80 218765_at Hs.33724 51092 NM_015996.1 NM_015996 CGI-40 protein 81218773_s_at Hs.279754 22921 NM_012228.1 NM_012228 pilin-liketranscription factor NM_016064 82 218853_s_at Hs.57549 56180 NM_019556.1NM_019556 hypothetical protein dJ473B4 83 219236_at Hs.235873 79957NM_024897.1 NM_024897 hypothetical protein FLJ22672 84 219329_s_atHs.9527 51374 NM_016085.1 NM_016085 apoptosis related protein APR-3NM_080592 apoptosis related protein APR-3 85 219694_at Hs.91165 54491NM_019018.1 NM_019018 hypothetical protein FLJ11127 86 219880_atHs.94037 64942 NM_022907.1 NM_022907 hypothetical protein FLJ23053 87220261_s_at Hs.5268 55146 NM_018106.1 NM_018106 hypothetical proteinFLJ10479 88 220387_s_at Hs.142245 11147 NM_007071.1 NM_007071 HERV-HLTR- associating 3 89 220520_s_at Hs.163629 54830 NM_017681.1 NM_017681hypothetical protein FLJ20130 90 221004_s_at Hs.111577 81618 NM_030926.1NM_030926 integral membrane protein 3 91 221449_s_at Hs.23047 81533NM_030790.1 NM_030790 hypothetical protein CDA08 92 221487_s_atHs.111680 2029 AF157510.1 NM_004436 endosulfine alpha 93 221958_s_atHs.250746 79971 AA775681 NM_024911 hypothetical protein FLJ23091 94222138_s_at AF158978.1 95 222209_s_at AK000684.1 96 55093_at Hs.8639254480 AA534198 97 56256_at Hs.33724 51092 AA150165 NM_015996 CGI-40protein

TABLE 2 Locus Ref Seq Affimetrix Unigene Link (NM_)/Gene Nr. ID ID IDBank ID Ref Seq ID + Annotation 1 200659_s_at Hs.75323 5245 NM_002634.2NM_002634 prohibitin 2 200685_at Hs.11482 9295 AU146237 NM_004768splicing factor, arginine/serine-rich 11 3 200691_s_at Hs.3069 3313BC000478.1 NM_004134 heat shock 70 kD protein 9B (mortalin-2) 4200692_s_at Hs.3069 3313 NM_004134.1 NM_004134 heat shock 70 kD protein9B (mortalin-2) 5 200831_s_at Hs.119597 6319 AA678241 NM_005063stearoyl-CoA desaturase (delta-9-desaturase) 6 200884_at Hs.173724 1152NM_001823.1 NM_001823 creatine kinase, brain 7 200987_x_at Hs.15297810197 AA758755 NM_005789 proteasome (prosome, macropain) activatorsubunit 3 (PA28 gamma; Ki) 8 201013_s_at Hs.117950 10606 AA902652NM_006452 phosphoribosylaminoimidazole carboxylase,phosphoribosylaminoribosylaminoimidazo 9 201016_at Hs.4310 1964 BE542684NM_001412 eukaryotic translation initiation factor 4C 10 201017_atHs.362973 BE542684 11 201231_s_at Hs.254105 2023 NM_001428.1 NM_001428enolase 1 NM_005945 12 201284_s_at Hs.78223 327 NM_001640.2 NM_001640N-acylaminoacyl- peptide hydrolase 13 201376_s_at Hs.808 3185 AI591354NM_004966 heterogeneous nuclear ribonucleoprotein F 14 201516_atHs.76244 6723 NM_003132.1 NM_003132 spermidine synthase 15 201600_atHs.7771 11331 NM_007273.1 NM_007273 B-cell associated protein 16201625_s_at Hs.56205 3638 BE300521 NM_005542 insulin induced gene 1 17201797_s_at Hs.159637 7407 NM_006295.1 NM_006295 valyl-tRNA synthetase 218 202115_s_at Hs.134200 26155 NM_015658.1 NM_015658 DKFZP564C186protein 19 202147_s_at Hs.7879 3475 NM_001550.1 NM_001550interferon-related developmental regulator 1 20 202159_at Hs.23111 2193NM_004461.1 NM_004461 phenylalanine-tRNA synthetase-like protein 21202462_s_at Hs.17585 9879 NM_014829.1 NM_014829 RNA helicase KIAA0801NM_016130 22 203119_at Hs.4253 79080 NM_024098.1 NM_024098 hypotheticalprotein MGC2574 23 203782_s_at Hs.153880 5442 NM_005035.1 NM_005035mitochondrial DNA- directed RNA polymerase precursor 24 204004_atHs.176090 5074 AI336206 NM_002583 apoptosis response protein 25206158_s_at Hs.2110 7555 NM_003418.1 NM_003418 zinc finger protein 9 26206424_at Hs.150595 1592 NM_000783.1 NM_000783 cytochrome P450,subfamily XXVIA, polypeptide 1, isoform 1 NM_057157 cytochrome P450,subfamily XXVIA, polypeptide 1, isoform 2 27 206445_s_at Hs.20521 3276NM_001536.1 NM_001536 HMT1 hnRNP methyltransferase-like 2 28 207001_x_atHs.75450 1831 NM_004089.1 NM_004089 delta sleep inducing peptide,immunoreactor 29 207397_s_at Hs.158309 3239 NM_000523.1 NM_000523 homeobox D13 30 207793_s_at Hs.37427 2035 NM_004437.1 NM_004437 erythrocytemembrane protein band 4.1 (elliptocytosis 1, RH-linked) 31 208152_s_atHs.169531 9188 NM_004728.1 NM_004728 DEAD/H (Asp-Glu- Ala-Asp/His) boxpolypeptide 21 32 208625_s_at Hs.211568 1981 AF104913.1 NM_004953eukaryotic translation initiation factor 4 gamma, 1 33 208763_s_atHs.75450 1831 AL110191.1 NM_004089 delta sleep inducing peptide,immunoreactor 34 208910_s_at Hs.78614 708 L04636.1 NM_001212 complementcomponent 1, q subcomponent binding protein precursor 35 208916_atHs.183556 6510 AF105230.1 NM_005628 solute carrier family 1 (neutralamino acid transporter), member 5 36 208920_at Hs.300741 6717 AV752215NM_003130 sorcin 37 209218_at Hs.71465 6713 AF098865.1 NM_003129squalene monooxygenase 38 210005_at Hs.82285 2618 D32051.1 NM_000819phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamidesynthetas 39 210337_s_at Hs.174140 47 U18197.1 NM_001096 ATP citratelyase 40 210347_s_at Hs.130881 53335 AF080216.1 NM_018014 B-cellCLL/lymphoma 11A, isoform 2 NM_022893 B-cell CLL/lymphoma 11A, isoform 1NM_138552 B-cell CLL/lymphoma 11A, isoform 4 NM_138553 B-cellCLL/lymphoma 11A, isoform 5 NM_138559 B-cell CLL/lymphoma 11A, isoform 341 211615_s_at Hs.182490 10128 M92439.1 NM_133259 leucine-rich PPR-motif containing 42 211708_s_at Hs.119597 6319 BC005807.1 NM_005063stearoyl-CoA desaturase (delta-9-desaturase) 43 211929_at Hs.24924710151 BE867771 NM_005758 heterogeneous nuclear ribonucleoprotein A3 44212009_s_at Hs.75612 10963 BF974063 NM_006819 stress-induced-phosphoprotein 1 (Hsp70/Hsp90- organizing protein) 45 212018_s_atAK025446.1 46 212174_at Hs.171811 204 AK023758.1 NM_001625 adenylatekinase 2 isoform a NM_013411 adenylate kinase 2 isoform b 47 212680_x_atHs.100623 26472 BE305165 NM_138689 phospholipase C, beta 3, neighbor 48213147_at Hs.110637 3206 NM_018951.1 NM_018951 homeo box A10 49213427_at Hs.115823 10799 NM_006638.1 NM_006638 ribonuclease P, 40 kDsubunit 50 213581_at Hs.351862 BF446180 51 213664_at Hs.91139 6505AW235061 NM_004170 solute carrier family 1 (neuronal/epithelial highaffinity glutamate transporter 52 213892_s_at Hs.28914 353 AA927724NM_000485 adenine phosphoribosyltransferase 53 214661_s_at Hs.1174878602 R06783 54 216212_s_at AJ010395 55 216397_s_at AK024840.1 56217027_x_at AC004941 57 217408_at AL050361.1 58 217985_s_at Hs.885811177 AA102574 NM_013448 bromodomain adjacent to zinc finger domain, 1A59 218081_at Hs.274422 54976 NM_017874.1 NM_017874 hypothetical proteinFLJ20550 60 218105_s_at Hs.279652 51073 NM_015956.1 NM_015956mitochondrial ribosomal protein L4 61 218112_at Hs.157160 65993NM_023936.1 NM_023936 mitochondrial ribosomal protein S34 62 218188_s_atHs.23410 26517 NM_012458.1 NM_012458 translocase of inner mitochondrialmembrane 13 homolog 63 218305_at Hs.61790 79711 NM_024658.1 NM_024658importin 4 64 218493_at Hs.15277 79622 NM_024571.1 NM_024571hypothetical protein FLJ22940 65 218680_x_at Hs.300954 25764 NM_016400.1NM_016400 Huntingtin interacting protein K 66 218889_at Hs.74899 64318NM_022451.1 NM_022451 AD24 protein 67 218893_at Hs.103833 79763NM_024710.1 NM_024710 hypothetical protein FLJ23469 68 219433_atHs.130732 54880 NM_017745.1 NM_017745 BCL-6 interacting corepressor,isoform 1 NM_020926 BCL-6 interacting corepressor, isoform, 2 69221691_x_at Hs.9614 4869 AB042278.1 NM_002520 nucleophosmin (nucleolarphosphoprotein B23, numatrin)

1. A method for detecting teneurin signalling, which method comprises:a) determining the presence of a cleaved teneurin product associatedwith teneurin signalling, wherein said cleaved teneurin productcomprises at least a portion of the cytoplasmic domain of teneurin andtargets to the cell nucleus; and b) correlating the presence and/oramount of said cleaved teneurin product with teneurin signalling.
 2. Amethod as claimed in claim 1, wherein said teneurin is teneurin-1,teneurin-2, teneurin-3 or teneurin-4.
 3. A method as claimed in of thepreceding claims wherein the cleaved teneurin product is formed inturnout cells.
 4. A method as claimed in claim 1 or 2, wherein thecleaved teneurin product is formed in neurons.
 5. A method as claimed inof the preceding claims further comprising providing a teneurin or afragment thereof comprising at least a portion of the N terminal domainof teneurin and at least a portion of the C-terminal domain of teneurin,and a cellular component that cleaves teneurin.
 6. A method as claimedin claim 5, wherein the teneurin is recombinant.
 7. A method as claimedin claim 6 wherein the cleaved teneurin product comprises a tag orlabel.
 8. A method as claimed in claim 7, wherein said determining step(b) comprises detecting said tag or label photometrically.
 9. A methodas claimed in claim 7, wherein said tag is selected from the groupconsisting of GFP, YFP, hemagluttanin, (Histidine)₇, a DNA bindingdomain.
 10. A method as claimed in claim 9 wherein said determining step(b) comprises allowing said DNA binding domain to bind to a nucleic acidcomprising regulatory sequences operably linked to a reporter gene anddetecting activity of said reporter gene.
 11. A method as claimed inclaim 10 wherein said DNA binding domain comprises a GAL4 DNA bindingdomain.
 12. A method as claimed in claim 9, wherein said tag is a DNAbinding domain and further comprises an NFκB domain.
 13. A method asclaimed in the preceding claim wherein said determining step comprisesdetermining the amount of said cleaved tenascin product.
 14. A method asclaimed in the preceding claims wherein the cleaved teneurin productregulates expression or activity of a cellular target.
 15. A method asclaimed in claim 14 further comprising detecting expression or activityof said cellular target.
 16. A method as claimed in claim 15 whereinsaid cellular target is PAL.
 17. A method as claimed in claim 15 whereinsaid cellular target is Zic.
 18. A method as claimed in claim 15 whereinsaid cellular target is ponsin.
 19. A method as claimed in claim 1,wherein the presence and/or amount of the cleaved teneurin product iscorrelated to a particular disease or condition.
 20. A method as claimedin claim 19, wherein said disease or condition is dependent on cellproliferation and/or neuronal differentiation.
 21. Use of a detectablecleaved teneurin product associated with teneurin signalling in a methodof diagnosis of a neuropathology or cell pathology affected by teneurinsignalling.
 22. A method for assessing the ability of an agent tomodulate teneurin signalling, comprising the steps of: (a) contactingteneurin with at least one agent; (b) detecting cleavage of saidteneurin by a cellular component associated with teneurin signalling inthe presence of said agent; and (c) correlating a difference in cleavageof said teneurin relative to when said agent is absent with anindication of the presence of an agent effective in modulating teneurinsignalling.
 23. A method as claimed in claim 22 wherein step (a) isperformed by perfusing a cell expressing recombinant teneurin with theagent.
 24. A method for assessing the ability of an agent to modulateteneurin-mediated signalling, comprising the steps of: (a) exposing acell to an agent; (b) detecting expression or activity of a generegulated by teneurin in said cell; and (c) correlating a change inexpression or activity of said gene with the presence of a modulator ofteneurin signalling.
 25. The use of an agent detected by a method ofclaim 22 for the manufacture of a medicament for the treatment orprophylactic treatment of a neuropathological condition.
 26. The use ofan agent detected by a method of claim 22 for the manufacture of amedicament for the treatment or prophylactic treatment of tumourigenesisor cancer.
 27. The use of a cleaved teneurin product associated withteneurin signalling, wherein said cleaved teneurin product comprises atleast a portion of the cytoplasmic domain of teneurin and targets to thecell nucleus; for the manufacture of a medicament for the treatment orprophylactic treatment of tumourigenesis or cancer.
 28. The use of acleaved teneurin product associated with teneurin signalling, whereinsaid cleaved teneurin product comprises at least a portion of thecytoplasmic domain of teneurin and targets to the cell nucleus; for themanufacture of a medicament for the treatment or prophylactic treatmentof a neuropathological condition.
 29. A method of treating an individualin need of treatment or prophylactic treatment of tumorogenesis, canceror a neuropathological condition, said method comprising administeringan effective amount of an agent identified by claim 22 sufficient toameliorate the symptoms of said individual.
 30. A method treating anindividual in need of treatment or prophylactic treatment oftumorogenesis, cancer or a neuropathological condition, said methodcomprising administering an effective amount of a cleaved teneurinproduct comprising at least a portion of the cytoplasmic domain ofteneurin, which targets to the cell nucleus, sufficient to amelioratethe symptoms of said individual.
 31. A composition comprising a cleavedteneurin product and a cellular target of the cleaved teneurin product.32. The composition of claim 31, wherein said cleaved teneurin productcomprises at least a portion of the cytoplasmic domain of teneurin andtargets to the cell nucleus.
 33. A composition as claimed in claim 31wherein said cellular target is PML.
 34. A composition as claimed inclaim 31 wherein said cellular target is Zic.
 35. A composition asclaimed in claim 31 wherein said cellular target is ponsin.
 36. Acomposition as claimed in claim 31 wherein said cellular target is myc.37. A composition as claimed in claim 31 wherein said cellular target isp53.
 38. A kit comprising a teneurin and a protease.